# Patent

## Title
Production of isobutanol in yeast mitochondria

## Abstract

Yeast cells with modified expression of certain enzyme activities in the mitochondria are described for isobutanol production. Modifications described provide an isobutanol biosynthesis pathway in the yeast mitochondria.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is related to and claims the benefit of priority of U.S. Provisional Application No. 61/114,073, filed Nov. 13, 2008, the entirety of which is herein incorporated by reference.


FIELD OF THE INVENTION
The invention relates to the field of microbiology. More specifically, recombinant yeast strains are disclosed that are engineered for isobutanol production in the mitochondria.
BACKGROUND OF THE INVENTION
Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Isobutanol is produced biologically as a by-product of yeast fermentation. It is a component of “fusel oil” that forms as a result of incomplete metabolism of amino acids by this group of fungi. Isobutanol is specifically produced from catabolism of L-valine in the cytoplasm. After the amine group of L-valine is harvested as a nitrogen source, the resulting α-keto acid is decarboxylated and reduced to isobutanol by enzymes of the so-called Ehrlich pathway (Dickinson et al., J. Biol. Chem. 273(40):25752-25756 (1998)). Yields of fusel oil and/or its components achieved during beverage fermentation are typically low. For example, the concentration of isobutanol produced in beer fermentation is reported to be less than 16 parts per million (Garcia et al., Process Biochemistry 29:303-309 (1994)). Addition of exogenous L-valine to the fermentation increases the yield of isobutanol, as described by Dickinson et al., supra, wherein it is reported that a yield of isobutanol of 3 g/L is obtained by providing L-valine at a concentration of 20 g/L in the fermentation. However, the use of valine as a feed-stock would be cost prohibitive for industrial scale isobutanol production.
Disclosed in US Patent Publication US20070092957 A1 is engineering of microorganisms for expression of several isobutanol biosynthetic pathways. Additionally Van Nedervelde et al (Proceedings of the Congress—European Brewery Convention (2003), 29th, 50/1-50/10) have demonstrated that deletions of the gene encoding the BAT1 mitochondrial protein in yeast resulted in strains having increased levels of higher alcohols. Similarly Nako et al (WO 2007032522) note that amyl alcohol and/or isobutanol and/or isoamyl acetate levels in yeast used for the production of alcoholic beverages may be altered via manipulation of the BAT1 and BAT2 genes. The art is silent with respect to the down regulation of other genes encoding proteins that are functional in the mitochondria for the enhanced production of isobutanol in yeast.
There is a need for attaining higher amounts of isobutanol through yeast fermentation without addition of valine or other isobutanol production intermediates.
SUMMARY OF THE INVENTION
Provided herein are recombinant yeast cells comprising a heterologous gene encoding a mitochondrial localized polypeptide having α-keto acid decarboxylase activity. In some embodiments, the α-keto acid decarboxylase activity is defined by the enzyme classification number EC 4.1.1.72.
In some embodiments, the cells produce isobutanol in the mitochondria. In some embodiments, the yeast cells comprise genes encoding mitochondrial localized polypeptides having ketol-acid reductoisomerase activity and dihydroxy-acid dehydratase activity which are overexpressed. In some embodiments, either one or both of the mitochondrial localized polypeptides having ketol-acid reductoisomerase activity and dihydroxy-acid dehydratase activity are heterologous to the yeast cell. In some embodiments, the ketol-acid reductoisomerase activity is defined by the enzyme classification number EC EC 1.1.1.86 and the dihydroxy-acid dehydratase activity is defined by the enzyme classification number EC 4.2.1.9. In some embodiments, the polypeptide having dihydroxy-acid dehydratase activity is a [4Fe-4S] or a [2Fe-2S] dihydroxy-acid dehydratase.
In some embodiments, the yeast cells comprise at least one gene encoding a mitochondrial localized polypeptide having acetolactate synthase activity which is overexpressed. In some embodiments, the at least one mitochondrial localized polypeptide having acetolactate synthase activity is heterologous to the yeast cell. In some embodiments, the acetolactate synthase activity is defined by the enzyme classification number EC 2.2.1.6.
Provided herein are recombinant yeast cells wherein the mitochondria is substantially devoid of a functional polypeptide having an enzyme activity selected from the group consisting of: threonine deaminase activity; isopropylmalate synthase activity, branched chain amino acid transaminase activity and pyruvate dehydrogenase activity.
In some embodiments, a) the threonine deaminase activity is defined by the enzyme classification number EC 4.3.1.19; b) the isopropylmalate synthase activity is defined by the enzyme classification number EC 2.3.3.13; c) the branched chain amino acid transaminase activity is defined by the enzyme classification number EC 2.6.1.42; and d) the pyruvate dehydrogenase activity is defined by the enzyme classification number EC 1.2.4.1. In some embodiments, the polypeptide having the pyruvate dehydrogenase activity is a multienzyme complex comprising proteins selected from the group consisting of: PDA1, PDA1, PDB1, LAT1, LPD1, and PDX1.
In some embodiments, the yeast cells comprise a gene encoding a polypeptide localized in the mitochondria having ATP-NAD (NADH) kinase activity which is over-expressed. In some embodiments, the ATP-NAD (NADH) kinase activity is defined by the enzyme classification number EC 2.7.1.86.
In some embodiments, the yeast cells are selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia. In some embodiments, the cells comprise reduced pyruvate decarboxylase activity.
In some embodiments, the yeast cell is a Saccharomyces and wherein; a) the polypeptide having ketol-acid reductoisomerase activity has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO 144; b) the polypeptide having dihydroxy-acid dehydratase activity has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO 552; c) the polypeptide having acetolactate synthase activity has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO:728; d) the polypeptide having threonine deaminase activity has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO2; e) the polypeptide having isopropylmalate synthase activity has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO 27; f) the polypeptide having branched chain amino acid transaminase activity has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO 15; wherein identity of polypeptides recited in parts (a)-(f) is based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
In some embodiments, the cell is a Saccharomyces and wherein the polypeptide having ATP-NAD (NADH) kinase activity has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO 720, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
Provided herein are methods of making a recombinant yeast cell for the production of isobutanol in the mitochondria comprising the steps of: a) providing a yeast comprising mitochondrial biosynthetic pathways for the production of isoleucine from threonine and production of isopropyl malate, valine, and acetyl-CoA from pyruvate wherein the pathway for the production of isopropyl malate, valine, and acetyl-CoA from pyruvate comprises genes encoding polypeptides having the following activities:

    
    
        i) ketol-acid reductoisomerase activity
        ii) dihydroxy-acid dehydratase activity
        iii) acetolactate synthase;
        iv) isopropylmalate synthase activity,
        v) branched chain amino acid transaminase activity;
        vi) pyruvate dehydrogenase activity; and
        vii) ATP-NAD (NADH) kinase activity;

and wherein the pathway for the production of isoleucine from threonine comprises at least one gene encoding a polypeptide having threonine deaminase activity;

    
    


b) introducing into the yeast of (a) at least one gene encoding a mitochondria localized polypeptide having α-keto acid decarboxylase activity;
c) increasing the expression of the polypeptides having ketol-acid reductoisomerase and dihydroxy-acid dehydratase activities; wherein isobutanol is produced in the mitochondria of the yeast cell. In some embodiments, the expression of the polypeptide having acetolactate synthase is increased. In some embodiments, the expression of the polypeptide having an ATP-NAD (NADH) kinase activity is increased.
In some embodiments at least one gene encoding a polypeptide having at least one of the following activities is down-regulated: a) an isopropylmalate synthase activity, b) a branched chain amino acid transaminase activity; c) a pyruvate dehydrogenase activity; and d) threonine deaminase activity. In some embodiments, the polypeptide having a ketol-acid reductoisomerase activity comprises a mitochondrial targeting signal sequence. In some embodiments, the polypeptide having a dihydroxy-acid dehydratase activity comprises a mitochondrial targeting signal sequence. In some embodiments, the polypeptide having a α-keto acid decarboxylase activity comprises a mitochondrial targeting signal sequence. In some embodiments, the polypeptide having a ATP-NAD (NADH) kinase activity comprises a mitochondrial targeting signal sequence.
Also provided are methods for making isobutanol comprising growing the yeast host cells provided herein under conditions whereby isobutanol is produced.



BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS
The invention can be more fully understood from the following detailed description, figures, and the accompanying sequence descriptions, which form a part of this application.
FIG. 1 shows branched chain amino acid biosynthetic pathways and an engineered isobutanol biosynthetic pathway in yeast mitochondria.



Table 8 is a table of the Profile HMM for dihydroxy-acid dehydratases based on enzymes with assayed function. Table 8 is submitted herewith electronically and is incorporated herein by reference.
The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.







TABLE 1







SEQ ID NOs of target proteins and encoding sequences for


reduction












SEQ ID
SEQ ID




NO:
NO:




nucleic
amino



Organism and gene name
acid
acid















Saccharomyces cerevisiae YJM789, Ilv1
1
2



Schizosaccharomyces pombe, Ilv1
735
3



Candida albicans SC5314, Ilv1
4
5



Candida glabrata, Ilv1
6
7



Kluyveromyces lactis, Ilv1
8
9



Yarrowia lipolytica strain CLIB122, Ilv1
10
11



Pichia stipitis CBS 6054, Ilv1
12
13



Saccharomyces cerevisiae, BAT1
14
15



Schizosaccharomycs pombe, BAT1
16
17



Candida albicans SC5314, BAT1
18
19



Kluyveromyces lactis, BAT1
20
21



Yarrowia lipolytica, BAT1
22
23



Pichia stipitis CBS 6054, BAT1
24
25



Saccharomyces cerevisiae, Leu4
26
27



Schizosaccharomycs pombe, Leu4
28
29



chromosome II



Schizosaccharomycs pombe,
30
31



Leu4, NP_596103.2



Candida albicans SC5314, Leu4
32
33



Candida albicans SC5314, Leu4
34
35



Candida albicans SC5314, Leu4
36
37



Candida albicans SC5314, Leu4
38
39



Candida glabrata, Leu4; XP_446653.1
40
41



Candida glabrata, Leu4; XP_446566.1
42
43



Kluyveromyces lactis, Leu4;
44
45



CAH00792.1



Kluyveromyces lactis, Leu4;
46
47



CAG98836.1



Yarrowia lipolytica, Leu4, CAA88928.1
48
49



Yarrowia lipolytica, Leu4
50
51



Pichia stipitis CBS 6054, Leu4,
52
53



XP_001387341.1



Pichia stipitis CBS 6054, Leu4,
54
55



XP_001384536.2



Saccharomyces cerevisiae, PDB1
56
57



Schizosaccharomycs pombe, PDB1
58
59



Candida albicans SC5314, PDB1
60
61



Kluyveromyces lactis, PDB1
62
63



Yarrowia lipolytica, PDB1
64
65



Pichia stipitis CBS 6054, PDB1
66
67



Saccharomyces cerevisiae, PDA1
68
69



Schizosaccharomycs pombe, PDA1
70
71



Candida albicans, PDA1
72
73



Kluyveromyces lactis, PDA1
74
75



Yarrowia lipolytica, PDA1 hypothetical
76
77



protein



Pichia stipitis, PDA1
78
79



Saccharomyces cerevisiae Lat1 pyruvate
729
730



dehydrogenase complex



Saccharomyces cerevisiae Lpd1
731
732



pyruvate dehydrogenase complex



Saccharomyces cerevisiae Pdx1
733
734



pyruvate dehydrogenase complex

















TABLE 2







SEQ ID NOs of target proteins and encoding sequences for


expression










SEQ ID
SEQ ID



NO:
NO:


Organism, gene name and/or enzyme
nucleic
amino


name
acid
acid





Lactococcus lactis, KivD (α-keto acid
123
124


decarboxylase)


Lactococcus lactis, KdcA (α-keto acid
125
126


decarboxylase)


Staphylococcus epidermis ATCC 12228,
127
128


Pdc (indole-3-pyruvate decarboxylase)


Bacillus cereus ATCC 14579, Pdc (indole-
129
130


3-pyruvate decarboxylase)


Clostridium acetobutyricum ATCC 824, Pdc
131
132


(pyruvate decarboxylase)


Pectobacterium atrosepticum SCRI1043,
133
134


Pdc (indole-3-pyruvate decarboxylase)


Serratia proteamaculans 568, Pdc (thiamine
135
136


pyrophosphate binding domain-containing


protein)


Escherichia coli, KARI (ketol-acid
137
138


reductoisomerase)


M maripaludis, KARI (ketol-acid
139
140


reductoisomerase)


Bacillus subtilis, KARI (ketol-acid
141
142


reductoisomerase)


Saccharomyces cerevisiae, KARI (ketol-
143
144


acid reductoisomerase)


Vibrio cholerae, KARI (ketol-acid
145
146


reductoisomerase)


Pseudomonas aeruginosa PAO1, IlvC
147
148


Pseudomonas fluorescens PF5, IlvC
149
150


Candida glabrata, KARI
151
152


Kluyveromyces lactis, KARI
153
154


Ashbya gossypii ATCC 10895, KARI
155
156


(ACL198Wp)


Pichia stipitis CBS 6054, KARI
157
158


(mitochondrial ketol-acid reductoisomerase)


Yarrowia lipolytica, KARI
159
160


Schizosaccharomyces pombe, KARI
161
162


(acetohydroxyacid reductoisomerase)


Bacillus subtilis, ALS (acetolactate
689
690


synthase)


Klebsiella pneumoniae, ALS (acetolactate
691
692


synthase)


Lactococcus lactis, ALS (acetolactate
693
694


synthase)


Staphylococcus aureus, ALS (acetolactate
695
696


synthase)


Listeria monocyto genes, ALS (acetolactate
697
698


synthase)


Streptococcus mutans, ALS (acetolactate
699
700


synthase)


Streptococcus thermophilus, ALS
701
702


(acetolactate synthase)


Vibrio angustum, ALS (acetolactate
703
704


synthase)


Bacillus cereus, ALS (acetolactate
705
706


synthase)


Saccharomyces cerevisiae, ALS
727
728


(acetolactate synthase)


Candida glabrata, ALS (acetolactate
707
708


synthase)


Kluyveromyces lactis, ALS (acetolactate
709
710


synthase)


Ashbya gossypii ATCC 10895, ALS
711
712


(acetolactate synthase)


Pichia stipitis CBS 6054, ALS (acetolactate
713
714


synthase)


Yarrowia lipolytica, ALS (acetolactate
715
716


synthase)


Aspergillus nidulans FGSC A4, ALS
717
718


(acetolactate synthase)


Saccharomyces cerevisiae, POS5 (NADH
719
720


kinase)


Candida glabrata, POS5 (NADH kinase)
721
722


Kluveromyces lactis, POS5 (NADH kinase)
723
724


Pichia stipitis CBS 6054, POS5 (NADH
725
726


kinase)
















TABLE 3







SEQ ID NOs of representative bacterial [2Fe—2S] DHAD


proteins and encoding sequences










SEQ ID
SEQ ID



NO:
NO:



nucleic
amino


Organism of derivation
acid
acid












Mycobacterium sp. MCS
163
164


Mycobacterium gilvum PYR-GCK
165
166


Mycobacterium smegmatis str. MC2 155
167
168


Mycobacterium vanbaalenii PYR-1
169
170


Nocardia farcinica IFM 10152
171
172


Rhodococcus sp. RHA1
173
174


Mycobacterium ulcerans Agy99
175
176


Mycobacterium avium subsp.
177
178


paratuberculosis K-10


Mycobacterium tuberculosis H37Ra
179
180


Mycobacterium leprae TN*
181
182


Kineococcus radiotolerans SRS30216
183
184


Janibacter sp. HTCC2649
185
186


Nocardioides sp. JS614
187
188


Renibacterium salmoninarum ATCC 33209
189
190


Arthrobacter aurescens TC1
191
192


Leifsonia xyli subsp. xyli str. CTCB07
193
194


marine actinobacterium PHSC20C1
195
196


Clavibacter michiganensis subsp.
197
198


michiganensis NCPPB 382


Saccharopolyspora erythraea NRRL 2338
199
200


Acidothermus cellulolyticus 11B
201
202


Corynebacterium efficiens YS-314
203
204


Brevibacterium linens BL2
205
206


Tropheryma whipplei TW08/27
207
208


Methylobacterium extorquens PA1
209
210


Methylobacterium nodulans ORS 2060
211
212


Rhodopseudomonas palustris BisB5
213
214


Rhodopseudomonas palustris BisB18
215
216


Bradyrhizobium sp. ORS278
217
218


Bradyrhizobium japonicum USDA 110
219
220


Fulvimarina pelagi HTCC2506
221
222


Aurantimonas sp. SI85-9A1
223
224


Hoeflea phototrophica DFL-43
225
226


Mesorhizobium loti MAFF303099
227
228


Mesorhizobium sp. BNC1
229
230


Parvibaculum lavamentivorans DS-1
231
232


Loktanella vestfoldensis SKA53
233
234


Roseobacter sp. CCS2
235
236


Dinoroseobacter shibae DFL 12
237
238


Roseovarius nubinhibens ISM
239
240


Sagittula stellata E-37
241
242


Roseobacter sp. AzwK-3b
243
244


Roseovarius sp. TM1035
245
246


Oceanicola batsensis HTCC2597
247
248


Oceanicola granulosus HTCC2516
249
250


Rhodobacterales bacterium HTCC2150
251
252


Paracoccus denitrificans PD1222
253
254


Oceanibulbus indolifex HEL-45
255
256


Sulfitobacter sp. EE-36
257
258


Roseobacter denitrificans OCh 114
259
260


Jannaschia sp. CCS1
261
262


Caulobacter sp. K31
263
264


Candidatus Pelagibacter ubique HTCC1062
265
266


Erythrobacter litoralis HTCC2594
267
268


Erythrobacter sp. NAP1
269
270


Comamonas testosterone KF-1
271
272


Sphingomonas wittichii RW1
273
274


Burkholderia xenovorans LB400
275
276


Burkholderia phytofirmans PsJN
277
278


Bordetella petrii DSM 12804
279
280


Bordetella bronchiseptica RB50
281
282


Bradyrhizobium sp. ORS278
283
284


Bradyrhizobium sp. BTAi1
285
286


Bradhyrhizobium japonicum
287
288


Sphingomonas wittichii RW1
289
290


Rhodobacterales bacterium HTCC2654
291
292


Solibacter usitatus Ellin6076
293
294


Roseiflexus sp. RS-1
295
296


Rubrobacter xylanophilus DSM 9941
927
298


Salinispora tropica CNB-440
299
300


Acidobacteria bacterium Ellin345
301
302


Thermus thermophilus HB27
303
304


Maricaulis maris MCS10
305
306


Parvularcula bermudensis HTCC2503
307
308


Oceanicaulis alexandrii HTCC2633
309
310


Plesiocystis pacifica SIR-1
311
312


Bacillus sp. NRRL B-14911
313
314


Oceanobacillus iheyensis HTE831
315
316


Staphylococcus saprophyticus subsp.
317
318


saprophyticus ATCC 15305


Bacillus selenitireducens MLS10
319
320


Streptococcus pneumoniae SP6-BS73
321
322


Streptococcus sanguinis SK36
323
324


Streptococcus thermophilus LMG 18311
325
326


Streptococcus suis 89/1591
327
328


Streptococcus mutans UA159
329
330


Leptospira borgpetersenii serovar Hardjo-
331
332


bovis L550


Candidatus Vesicomyosocius okutanii HA
333
334


Candidatus Ruthia magnifica str. Cm
335
336


(Calyptogena magnifica)


Methylococcus capsulatus str. Bath
337
338


uncultured marine bacterium EB80_02D08
339
340


uncultured marine gamma proteobacterium
341
342


EBAC31A08


uncultured marine gamma proteobacterium
343
344


EBAC20E09


uncultured gamma proteobacterium
345
346


eBACHOT4E07


Alcanivorax borkumensis SK2
347
348


Chromohalobacter salexigens DSM 3043
349
350


Marinobacter algicola DG893
351
352


Marinobacter aquaeolei VT8
353
354


Marinobacter sp. ELB17
355
356


Pseudoalteromonas haloplanktis TAC125
357
358


Acinetobacter sp. ADP1
359
360


Opitutaceae bacterium TAV2
361
362


Flavobacterium sp. MED217
363
264


Cellulophaga sp. MED134
365
366


Kordia algicida OT-1
367
368


Flavobacteriales bacterium ALC-1
369
370


Psychroflexus torquis ATCC 700755
371
372


Flavobacteriales bacterium HTCC2170
373
374


unidentified eubacterium SCB49
375
376


Gramella forsetii KT0803
377
378


Robiginitalea biformata HTCC2501
379
380


Tenacibaculum sp. MED152
381
382


Polaribacter irgensii 23-P
383
384


Pedobacter sp. BAL39
385
386


Flavobacteria bacterium BAL38
387
388


Flavobacterium psychrophilum JIP02/86
389
390


Flavobacterium johnsoniae UW101
391
392


Lactococcus lactis subsp. cremoris SK11
393
394


Psychromonas ingrahamii 37
395
396


Microscilla marina ATCC 23134
397
398


Cytophaga hutchinsonii ATCC 33406
399
400


Rhodopirellula baltica SH 1
401
402


Blastopirellula marina DSM 3645
403
404


Planctomyces maris DSM 8797
405
406


Algoriphagus sp. PR1
407
408


Candidatus Sulcia muelleri str. Hc
409
410


(Homalodisca coagulata)


Candidatus Carsonella ruddii PV
411
412


Synechococcus sp. RS9916
413
414


Synechococcus sp. WH 7803
415
416


Synechococcus sp. CC9311
417
418


Synechococcus sp. CC9605
419
420


Synechococcus sp. WH 8102
421
422


Synechococcus sp. BL107
423
424


Synechococcus sp. RCC307
425
426


Synechococcus sp. RS9917
427
428


Synechococcus sp. WH 5701
429
430


Prochlorococcus marinus str. MIT 9313
431
432


Prochlorococcus marinus str. NATL2A
433
434


Prochlorococcus marinus str. MIT 9215
435
436


Prochlorococcus marinus str. AS9601
437
438


Prochlorococcus marinus str. MIT 9515
439
440


Prochlorococcus marinus subsp. pastoris str.
441
442


CCMP1986


Prochlorococcus marinus str. MIT 9211
443
444


Prochlorococcus marinus subsp. marinus str.
445
446


CCMP1375


Nodularia spumigena CCY9414
447
448


Nostoc punctiforme PCC 73102
449
450


Nostoc sp. PCC 7120
451
452


Trichodesmium erythraeum IMS101
453
454


Acaryochloris marina MBIC11017
455
456


Lyngbya sp. PCC 8106
457
458


Synechocystis sp. PCC 6803
459
460


Cyanothece sp. CCY0110
461
462


Thermosynechococcus elongatus BP-1
463
464


Synechococcus sp. JA-2-3B′a(2-13)
465
466


Gloeobacter violaceus PCC 7421
467
468


Nitrosomonas eutropha C91
469
470


Nitrosomonas europaea ATCC 19718
471
472


Nitrosospira multiformis ATCC 25196
473
474


Chloroflexus aggregans DSM 9485
475
476


Leptospirillum sp. Group II UBA
477
478


Leptospirillum sp. Group II UBA
479
480


Halorhodospira halophila SL1
481
482


Nitrococcus mobilis Nb-231
483
484


Alkalilimnicola ehrlichei MLHE-1
485
486


Deinococcus geothermalis DSM 11300
487
488


Polynucleobacter sp. QLW-P1DMWA-1
489
490


Polynucleobacter necessarius STIR1
491
492


Azoarcus sp. EbN1
493
494


Burkholderia phymatum STM815
495
496


Burkholderia xenovorans LB400
497
498


Burkholderia multivorans ATCC 17616
499
500


Burkholderia cenocepacia PC184
501
502


Burkholderia mallei GB8 horse 4
503
504


Ralstonia eutropha JMP134
505
506


Ralstonia metallidurans CH34
507
508


Ralstonia solanacearum UW551
509
510


Ralstonia pickettii 12J
511
512


Limnobacter sp. MED105
513
514


Herminiimonas arsenicoxydans
515
516


Bordetella parapertussis
517
518


Bordetella petrii DSM 12804
519
520


Polaromonas sp. JS666
521
522


Polaromonas naphthalenivorans CJ2
523
524


Rhodoferax ferrireducens T118
525
526


Verminephrobacter eiseniae EF01-2
527
528


Acidovorax sp. JS42
529
530


Delftia acidovorans SPH-1
531
532


Methylibium petroleiphilum PM1
533
534


gamma proteobacterium KT 71
535
536


Tremblaya princeps
537
538


Blastopirellula marina DSM 3645
539
540


Planctomyces maris DSM 8797
541
542


Microcystis aeruginosa PCC 7806
543
544


Salinibacter ruber DSM 13855
545
546


Methylobacterium chloromethanicum
547
548
















TABLE 4







SEQ ID NOs of representative fungal and plant [2Fe—2S]


DHAD proteins and encoding sequences












SEQ ID





NO:
SEQ ID NO:



Description
Nucleic acid
Peptide















Schizosaccharomyces pombe
549
550



ILV3



Saccharomyces cerevisiae ILV3
551
552



Kluyveromyces lactis ILV3
553
554



Candida albicans SC5314 ILV3
555
556



Pichia stipitis CBS 6054 ILV3
557
558



Yarrowia lipolytica ILV3
559
560



Candida galbrata CBS 138 ILV3
561
562



Chlamydomonas reinhardtii
563
564



Ostreococcus lucimarinus
565
566



CCE9901



Vitis vinifera (CAO71581.1)
567
568



Vitis vinifera
569
570



(CAN67446.1)



Arabidopsis thaliana
571
572



Oryza sativa (indica cultivar-
573
574



group)



Physcomitrella patens subsp.
575
576



patens



Chaetomium globosum CBS
577
578



148.51



Neurospora crassa OR74A
579
580



Magnaporthe grisea 70-15
581
582



Gibberella zeae PH-1
583
584



Aspergillus niger
585
586



Neosartorya fischeri NRRL 181
587
588



(XP_001266525.1)



Neosartorya fischeri NRRL 181
589
590



(XP_001262996.1)



Aspergillus niger (An03g04520)
591
592



Aspergillus niger (An14g03280)
593
594



Aspergillus terreus NIH2624
595
596



Aspergillus clavatus NRRL 1
597
598



Aspergillus nidulans FGSC A4
599
600



Aspergillus oryzae
601
602



Ajellomyces capsulatus NAm1
603
604



Coccidioides immitis RS
605
606



Botryotinia fuckeliana B05.10
607
608



Phaeosphaeria nodorum SN15
609
610



Pichia guilliermondii ATCC 6260
611
612



Debaryomyces hansenii CBS767
613
614



Lodderomyces elongisporus
615
616



NRRL YB-4239



Vanderwaltozyma polyspora DSM
617
618



70294



Ashbya gossypii ATCC 10895
619
620



Laccaria bicolor S238N-H82
621
622



Coprinopsis cinerea
623
624



okayama 7#130



Cryptococcus neoformans var.
625
626



neoformans JEC21



Ustilago maydis 521
627
628



Malassezia globosa CBS 7966
629
630



Aspergillus clavatus NRRL 1
631
632



Neosartorya fischeri NRRL 181
633
634



(Putative)



Aspergillus oryzae
635
636



Aspergillus niger (An18g04160)
637
638



Aspergillus terreus NIH2624
639
640



Coccidioides immitis RS
641
642



(CIMG_04591)



Paracoccidioides brasiliensis
643
644



Phaeosphaeria nodorum SN15
645
646



Gibberella zeae PH-1
647
648



Neurospora crassa OR74A
649
650



Coprinopsis cinerea okayama
651
652



7#130



Laccaria bicolor S238N-H82
653
654



Ustilago maydis 521
655
656

















TABLE 5







SEQ ID NOs of representative [4Fe—4S] DHAD proteins and


encoding sequences










SEQ ID
SEQ ID



NO: nucleic
NO: amino


Organism
acid
acid





Escherichia coli str. K-12 substr.
657
658


MG1655


Bacillus subtilis subsp. subtilis str. 168
659
660


Agrobacterium tumefaciens str. C58
661
662


Burkholderia cenocepacia MC0-3
663
664


Psychrobacter cryohalolentis K5
665
666


Psychromonas sp. CNPT3
667
668


Deinococcus radiodurans R1
669
670


Wolinella succinogenes DSM 1740
671
672


Zymomonas mobilis subsp. mobilis ZM4
673
674


Clostridium acetobutylicum ATCC 824
675
676


Clostridium beijerinckii NCIMB 8052
677
678


Pseudomonas fluorescens Pf-5
679
680


Methanococcus maripaludis C7
681
682


Methanococcus aeolicus Nankai-3
683
684


Vibrio fischeri ATCC 700601 (ES114)
685
686


Shewanella oneidensis MR-1 ATCC
687
688


700550
















TABLE 6







SEQ ID NOs of mitochondrial targeting signals and their


encoding sequences












SEQ ID
SEQ ID




NO:
NO:




nucleic
amino



Organism and gene
acid
acid







Saccharomyces cerevisiae, cytochrome C
116
117



oxidase (COX) subunit IV mitochondrial



targeting sequence



Saccharomyces cerevisiae, mitochondrial
119
120



targeting sequence (MTS) from the CDC9



DNA ligase gene



Saccharomyces cerevisiae, mitochondrial
121
122



targeting sequence (MTS) from the ATP2



ATP synthase

















TABLE 7







SEQ ID NOs of primers and vectors













SEQ ID NO:



Organism and gene name
Description
nucleic acid















pRS423::FBAp (pNY13)
vector
80



ILV5-R(Xho)
primer
81



ILV5-F(XhoPstAsc)
primer
82



pRS426::GPM-kivD
vector
83



112590-80A
primer
84



112590-80D
primer
85



N133SeqR4
primer
86



112590-91A
primer
87



112590-91B
primer
88



112590-88A
primer
89



112590-88B
primer
90



112590-88C
primer
91



112590-88D
primer
92



pUC19-URA3r
vector
93



112590-97A
primer
94



112590-97B
primer
95



112590-49E
primer
96



112590-97C
primer
97



112590-108A
primer
98



112590-108B
primer
99



112590-108C
primer
100



112590-108D
primer
101



112590-108E
primer
102



112590-108F
primer
103



BAT1 check
primer
104



112590-118A
primer
105



112590-118B
primer
106



pRS426::GAL1p-alsS
vector
107



112590-118C
primer
108



112590-118D
primer
109



112590-118E
primer
110



112590-118F
primer
111



112590-118G
primer
112

















TABLE 14







Pyruvate decarboxylase sequences












SEQ ID
SEQ ID




NO:
NO:




nucleic
amino



Organism and gene name
acid
acid







PDC1 pyruvate decarboxylase
728
729



from Saccharomyces cerevisiae



PDC5 pyruvate decarboxylase
730
731



from Saccharomyces cerevisiae



PDC6 pyruvate decarboxylase
732
733



from Saccharomyces cerevisiae



pyruvate decarboxylase from
734
735



Candida glabrata



PDC1 pyruvate decarboxylase
736
737



from Pichia stipitis



PDC2 pyruvate decarboxylase
738
739



from Pichia stipitis



pyruvate decarboxylase from
740
741



Kluyveromyces lactis



pyruvate decarboxylase from
742
743



Yarrowia lipolytica



pyruvate decarboxylase from
744
745



Schizosaccharomyces pombe










SEQ ID NO:113 is the nucleotide sequence of the GPM promoter from Saccharomyces cerevisiae. 
SEQ ID NO:114 is the nucleotide sequence of the kivD coding region from Lactococcus lactis that was codon optimized for expression in E. coli. 
SEQ ID NO: 115 is the nucleotide sequence of the ADH1 terminator from Saccharomyces cerevisiae. 
SEQ ID NO: 118 is the nucleotide sequence of the coding region of ILV3 from Kluyveromyces lactis that was codon optimized for expression in Saccharomyces cerevisiae. 
DETAILED DESCRIPTION OF THE INVENTION
Provided herein are recombinant yeast host cells having isobutanol production in the mitochondria. The disclosed yeast host cells may be used to produce isobutanol to provide an alternative to fossil fuels.
Provided herein are recombinant yeast cells engineered to have α-keto acid decarboxylase activity in the mitochondria in addition to having overexpressed ketol-acid reductoisomerase and dihydroxy-acid dehydratase activities in the mitochondria. These recombinant yeast cells have an isobutanol biosynthetic pathway in the mitochondria and produce isobutanol. The present recombinant yeast cells may have in addition overexpressed ATP-NAD (NADH) kinase activity in the mitochondria. The present recombinant yeast cells may have in addition reduced activity of threonine deaminase and isopropylmalate synthase in the mitochondria. Still further, the present recombinant yeast cells may have reduced pyruvate dehydrogenase activity in the mitochondria, and optionally reduced activity of branched chain amino acid transaminase in the mitochondria. These yeast cells produce increased amounts of isobutanol as compared to yeast cells without mitochondrial α-keto acid decarboxylase activity and with natural levels of the other enzyme activities stated above. Isobutanol is valuable as a fuel or fuel additive to reduce demand for fossil fuels.
The following abbreviations and definitions will be used for the interpretation of the specification and the claims.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.
The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.
As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value
The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.
The term “carbon substrate” or “fermentable carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention.
The terms “acetohydroxy acid isomeroreductase” and “ketol-acid reductoisomerase” and “acetohydroxy acid reductoisomerase” may be used interchangeably and refer the enzyme having the EC number, EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego). Ketol-acid reductoisomerase (KARI) catalyzes the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate. These enzymes are available from a number of sources, including, but not limited to E. coli (DNA: SEQ ID NO:137; protein SEQ ID NO:138), M. maripaludis (DNA: SEQ ID NO:139; protein SEQ ID NO:140), B. subtilis (DNA: SEQ ID NO:141; protein SEQ ID NO:142), and S. cerevisiae (DNA: SEQ ID NO:143; protein SEQ ID NO:144). In yeast the gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The term “α-keto acid decarboxylase” refers to an enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde (also called isobutanal) and CO2. Alpha-keto acid decarboxylase enzymes, also called 2-oxoacid decarboxylases, are known by the EC number, EC 4.1.1.72. Examples include KivD (DNA SEQ ID NO:123; protein SEQ ID NO:124) and KdcA (DNA SEQ ID NO:125; protein SEQ ID NO:126), both from Lactococcus lactis. Pyruvate decarboxylases have the ability to use the α-ketoisovalerate substrate, but prefer pyruvate as a substrate. Engineering of any of these enzymes for higher conversion of the α-ketoisovalerate substrate would make them useful to the pathway described herein. In yeast the gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The terms “acetolactate synthase” and “acetolactate synthetase” may be used intechangeably and refer to an enzyme that catalyzes the conversion of pyruvate to acetolactate and CO2. Preferred acetolactate synthases are known by the EC number, EC 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (DNA: SEQ ID NO:689; protein: SEQ ID NO:690), Klebsiella pneumoniae (DNA: SEQ ID NO:691; protein: SEQ ID NO:692), and Lactococcus lactis (DNA: SEQ ID NO:693; protein: SEQ ID NO:694). The gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The term “acetohydroxy acid dehydratase” and “dihydroxy acid dehydratase” refer to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate. Preferred dihydroxy acid dehydratases are known by the EC number 4.2.1.9. These enzymes are available from a vast array of microorganisms, including, but not limited to, E. coli (DNA SEQ ID NO:657, protein SEQ ID NO:658), S. cerevisiae (DNA SEQ ID NO:551, protein SEQ ID NO:552), M. maripaludis (DNA SEQ ID NO:681, protein SEQ ID NO:682), and B. subtilis (DNA SEQ ID NO:659, protein SEQ ID NO:660). The gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The term “threonine deaminase refers to an enzyme having the EC number EC 4.3.1.19 (Enzyme Nomenclature 1992, Academic Press, San Diego). Threonine deaminase catalyzes the reaction of threonine to 2-oxobutanoate. This is an enzyme involved in branched chain amino acid biosynthesis, specifically of isoleucine. The gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The term “isopropylmalate synthase” refers to an enzyme having the EC number, EC 2.3.3.13 (Enzyme Nomenclature 1992, Academic Press, San Diego). Isopropylmalate synthase catalyzes the reaction of alpha-ketoisovalerate to isopropyl malate. This is an enzyme involved in branched chain amino acid biosynthesis, specifically of leucine. The gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The term “branched chain amino acid transaminase” refers to an enzyme having the EC number, EC 2.6.1.42 (Enzyme Nomenclature 1992, Academic Press, San Diego). Branched chain amino acid transaminase catalyzes the reaction of alpha-ketoisovalerate to valine. This is an enzyme involved in branched chain amino acid biosynthesis, specifically of valine. The gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The term “NADH kinase” refers to an enzyme having the EC number, EC 2.7.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego). NADH kinase catalyzes the reaction of: ATP+NADH=ADP+NADPH. The gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The term “pyruvate dehydrogenase” refers to an activity provided by a multienzyme complex that includes proteins PDA1, PDB1, LAT1, LPD1, and PDX1. PDA1 and PDB1 are E1α and E1β subunits of pyruvate dehydrogenase activity which has EC number EC 1.2.4.1. LAT1 is dihydrolipoyllysine-residue acetyltransferase, also called dihydrolopoyl transacetylase, which has EC number EC 2.3.1.12. LPD1 is dihydrolipoyl dehydrogenase which has EC number EC 1.8.1.4. Pyruvate dehydrogenase activity catalyzes the reaction of pyruvate to acetyl-CoA. The gene encoding this enzyme is localized in the yeast nucleus however the encoded protein is transported to the mitochondria where it is metabolically active.
The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign gene” or “heterologous gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. “Heterologous gene” includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. Also foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
As used herein the term “coding region” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
The term “overexpression”, as used herein, refers to expression at a level that is above the expression level found in a cell prior to genetic manipulation.
As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid” and “vector” as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.
As used herein, an “isolated nucleic acid fragment” or “isolated nucleic acid molecule” will be used interchangeably and will mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
A nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular fungal proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: N.J. (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).
Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992)) and found in the MegAlignTM v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.
It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100% may be useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional methods used here are in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).
Isobutanol Biosynthesis Pathway in Yeast Mitochondria
Disclosed herein are yeast cells with improved isobutanol production due to engineering of an isobutanol biosynthetic pathway in the mitochondria of yeast. This engineering may be performed in any type of yeast cell that is amenable to genetic engineering methods. Suitable yeasts include, but are not limited to, Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia. Suitable strains include, but are not limited to, Saccharomyces cerevisiae Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata, Candida albicans, Pichia stipitis and Yarrowia lipolytica. 
Applicants have found that by expressing α-keto acid decarboxylase activity in yeast mitochondria, along with increasing expression of endogenous enzymes in mitochondrial branched chain amino acid biosynthetic pathways, isobutanol is made in increased amounts from a mitochondrial isobutanol biosynthetic pathway. Applicants found that by introducing α-keto acid decarboxylase activity into the mitochondria and overexpressing mitochondrial ketol-acid reductoisomerase and dihydroxy-acid dehydratase activities, about a seven-fold increase in isobutanol production was achieved. Further a 13 fold increase in isobutanol production was achieved when these modifications were made in a yeast strain where mitochondrial threonine deaminase and isopropylmalate synthase activities had been eliminated. Additional reduction of pyruvate dehydrogenase and branched chain amino acid transaminase activities in the mitochondria resulted in over fifteen-fold increase in isobutanol production.
Mitochondrial biosynthetic pathways for natural branched chain amino acid biosynthesis, and for engineered isobutanol biosynthesis, are shown in the diagram in FIG. 1. The following enzymes are encoded by the genes labeled as steps (arrows) in the pathways in FIG. 1:
ILV1: threonine deaminase
ILV2: acetolactate synthase (ALS)
ILV3: dihydroxy-acid dehydratase (DHAD)
ILV5: acetohydroxy acid reductoisomerase, also called ketol-acid reductoisomerase (KARI)
KivD: α-keto acid decarboxylase
ADH: alcohol dehydrogenase
BAT1: branched chain amino acid aminotransferase
BAT2: branched chain amino acid transaminase
LEU4: isopropylmalate synthase
PDA1*: refers to the complex including the components:

    
    
        PDA1: pyruvate dehydrogenase E1α subunit
        PDB1: pyruvate dehydrogenase E1β subunit
        LAT1: dihydrolipoyllysine-residue acetyltransferase
        LPD1: dihydrolipoyl dehydrogenase
        PDX1: protein X
    
    


POS5: mitochondrial NADH kinase
Threonine deaminase, ALS, KARI, DHAD and BAT1 enzyme activities in the mitochondria form a biosynthetic pathway from threonine to isoleucine. ALS, KARI, DHAD and BAT1 enzyme activities in the mitochondria form a biosynthetic pathway from pyruvate to valine, with α-ketoisovalerate as an intermediate. Alpha-ketoisovalerate that is synthesized in yeast mitochondria is also transported to the cytosol where cytoplasmic branched chain amino acid transaminase (BAT2) activity converts it to valine. An isobutanol biosynthetic pathway that includes ALS, KARI, DHAD, KivD and alcohol dehydrogenase is disclosed in US Patent Publication #US20070092957 A1. To provide an isobutanol biosynthetic pathway using α-ketoisovalerate produced in the mitochondria, relying on endogenous alcohol dehydrogenase activity, applicants expressed KivD in either the cytosol or mitochondria in yeast with increased mitochondrial expression of KARI and DHAD. Applicants found that isobutanol production was more than doubled when KivD activity was present in the mitochondria as compared to being in the cytosol.
Endogenous alcohol dehydrogenase activity may be provided by any enzyme, and preferably by ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7. Endogenous alcohol dehydrogenase activity is provided in both the mitochondria and the cytosol since ADH3 is localized in the mitochondria while ADH1, ADH2, and ADH4-7 are in the cytosol. If desired for increased butanol production, ADH activity may be increased by increasing expression of any of these enzymes. Preferred is overexpression in both the cytosol and the mitochondria. Cytosolic enzymes may be targeted to the mitochondria as described below for α-keto acid decarboxylase. Chimeric genes, vectors, transformation and expression of these genes may be achieved as described below.
Mitochondrial Localized α-Ketoisovalerate Decarboxylase
In the present disclosure yeast cells are engineered to express α-keto acid decarboxylase activity that is localized in the mitochondria. The skilled person will appreciate that polypeptides having α-keto acid decarboxylase activity isolated from any source will be useful in the present invention. Some examples of suitable α-keto acid decarboxylase enzymes, as described in the definitions above, are KivD (DNA SEQ ID NO:123; protein SEQ ID NO:124) and KdcA (DNA SEQ ID NO:125; protein SEQ ID NO:126), both from Lactococcus lactis. Additional proteins that have at least about 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to KivD or KdcA such as pyruvate decarboxylases from Staphylococcus epidermidis (DNA SEQ ID NO:127; protein SEQ ID NO:128), Bacillus cereus (DNA SEQ ID NO:129; protein SEQ ID NO:130), Clostridium acetobutyricum (DNA SEQ ID NO:131; protein SEQ ID NO:132), Pectobacterium atrosepticum (DNA SEQ ID NO:133; protein SEQ ID NO:134), and Serratia proteamaculans (DNA SEQ ID NO:135; protein SEQ ID NO:136), and that have α-keto acid decarboxylase activity, may be used in the present yeast cells. Typically BLAST (described above) searching of publicly available databases with known α-keto acid decarboxylase amino acid sequences, such as those provided herein, is used to identify additional α-keto acid decarboxylases, and their encoding sequences, that may be used in the present strains. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
In addition to using protein or coding region sequence and bioinformatics methods to identify additional α-keto acid decarboxylases, the sequences described herein or those recited in the art may be used to experimentally identify other homologs in nature. For example each of the α-keto acid decarboxylase encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.
For example, genes encoding similar proteins or polypeptides to the α-keto acid decarboxylase encoding genes described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired organism using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the disclosed nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments by hybridization under conditions of appropriate stringency.
Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).
Generally two short segments of the described sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the described nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding microbial genes.
Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (e.g., BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
Alternatively, the described α-keto acid decarboxylase encoding sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).
Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).
Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
For expression of α-keto acid decarboxylase, a coding region for an α-keto acid decarboxylase enzyme may be introduced into the mitochondrial genome as part of a chimeric gene for expression such that it is directly expressed in the mitochondrion. For yeast mitochondrial transformation DNA may be delivered to the mitochondrial matrix by high-velocity bombardment of yeast cells with tungsten microprojectiles carrying DNA for mitochondrial genome integration. Several high-velocity microprojectile bombardment devices are commercially available, and these are powered by gunpowder charge or compressed gas.
More typically, to obtain mitochondrial enzyme activity a mitochondrial targeting signal encoding sequence is operably linked to an α-keto acid decarboxylase coding region in a chimeric gene so that the expressed protein is translocated from the cytosol into the mitochondria. Any mitochondrial targeting signal sequence that is able to direct transport of a protein to the yeast mitochondrion may be used. Examples include mitochondrial targeting signal sequences that are present on proteins that are naturally transported to the mitochondrion in yeast such as KARI and DHAD. Some typically used mitochondrial targeting signals include those from S. cerevisiae cytochrome C oxidase (COX) subunit IV (coding region SEQ ID NO:116; amino acid SEQ ID NO:117; Hurt et al. EMBO J. (1984) 3(13):3149-56), S. cerevisiae CDC9 DNA ligase (coding region SEQ ID NO:119; amino acid SEQ ID NO:120: Donahue et al., (2001) Nucleic Acids Res. 29:1582-1589), and S. cerevisiae ATP2 ATP synthase (coding region SEQ ID NO:121; amino acid SEQ ID NO:122: Margeot et al, (2002) EMBO J. 21:6893-6904).
Expression of α-keto acid decarboxylase is achieved by transforming with a gene comprising a sequence encoding an α-keto acid decarboxylase protein. Methods for gene expression in yeasts are known in the art (see for example Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Expression of genes in yeast typically requires a promoter, operably linked to a coding region of interest, and a transcriptional terminator. A number of yeast promoters can be used in constructing expression cassettes for genes encoding an α-keto acid decarboxylase, including, but not limited to constitutive promoters FBA, GPD, ADH1, and GPM, and the inducible promoters GAL1, GAL10, and CUP1. Suitable transcriptional terminators include, but are not limited to FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1, and ADH1.
Suitable promoters, transcriptional terminators, and α-keto acid decarboxylase coding regions may be cloned into E. coli-yeast shuttle vectors, and transformed into yeast cells as described in Examples 2-4. These vectors allow strain propagation in both E. coli and yeast strains. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. Typically used plasmids in yeast are shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.), which contain an E. coli replication origin (e.g., pMB1), a yeast 2μ origin of replication, and a marker for nutritional selection. The selection markers for these four vectors are His3 (vector pRS423), Trp1 (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). Construction of expression vectors with a chimeric gene encoding an α-keto acid decarboxylase may be performed by either standard molecular cloning techniques in E. coli or by the gap repair recombination method in yeast.
The gap repair cloning approach takes advantage of the highly efficient homologous recombination in yeast. Typically, a yeast vector DNA is digested (e.g., in its multiple cloning site) to create a “gap” in its sequence. A number of insert DNAs of interest are generated that contain a ≧21 bp sequence at both the 5′ and the 3′ ends that sequentially overlap with each other, and with the 5′ and 3′ terminus of the vector DNA. For example, to construct a yeast expression vector for “Gene X’, a yeast promoter and a yeast terminator are selected for the expression cassette. The promoter and terminator are amplified from the yeast genomic DNA, and Gene X is either PCR amplified from its source organism or obtained from a cloning vector comprising Gene X sequence. There is at least a 21 bp overlapping sequence between the 5′ end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3′ end of the linearized vector. The “gapped” vector and the insert DNAs are then co-transformed into a yeast strain and plated on the medium containing the appropriate compound mixtures that allow complementation of the nutritional selection markers on the plasmids. The presence of correct insert combinations can be confirmed by PCR mapping using plasmid DNA prepared from the selected cells. The plasmid DNA isolated from yeast (usually low in concentration) can then be transformed into an E. coli strain, e.g. TOP10, followed by mini preps and restriction mapping to further verify the plasmid construct. Finally the construct can be verified by sequence analysis.
Like the gap repair technique, integration into the yeast genome also takes advantage of the homologous recombination system in yeast. Typically, a cassette containing a coding region plus control elements (promoter and terminator) and auxotrophic marker is PCR-amplified with a high-fidelity DNA polymerase using primers that hybridize to the cassette and contain 40-70 base pairs of sequence homology to the regions 5′ and 3′ of the genomic area where insertion is desired. The PCR product is then transformed into yeast and plated on medium containing the appropriate compound mixtures that allow selection for the integrated auxotrophic marker. For example, to integrate “Gene X” into chromosomal location “Y”, the promoter-coding regionX-terminator construct is PCR amplified from a plasmid DNA construct and joined to an autotrophic marker (such as URA3) by either SOE PCR or by common restriction digests and cloning. The full cassette, containing the promoter-coding regionX-terminator-URA3 region, is PCR amplified with primer sequences that contain 40-70 bp of homology to the regions 5′ and 3′ of location “Y” on the yeast chromosome. The PCR product is transformed into yeast and selected on growth media lacking uracil. Transformants can be verified either by colony PCR or by direct sequencing of chromosomal DNA.
Overexpression of α-Ketoisovalerate Substrate Production in the Mitochondria
In the present yeast cells expression of ketol-acid reductoisomerase and dihydroxy-acid dehydratase activities in the mitochondria is increased to increase mitochondrial biosynthesis of the α-ketoisovalerate substrate of the introduced mitochondrial α-keto acid decarboxylase activity disclosed above. Ketol-acid reductoisomerase (KARI) and dihydroxy-acid dehydratase (DHAD) are endogenous activities in yeast mitochondria. These enzyme activities may be overexpressed by engineering the cells for increased expression of the endogenous coding regions or by introducing genes for expression of heterologous coding regions for these enzymes. Genes for expression of KARI or DHAD may include sequences encoding mitochondrial targeting signals for translocation of the proteins from the cytosol to the mitochondria, or may be transformed into the yeast mitochondrial genome as described for mitochondrial α-keto acid decarboxylase expression.
Yeast KARI is encoded by an ILV5 gene. ILV5 encodes KARI and includes a mitochondrial targeting signal for translocation of the protein to the mitochondria. A KARI is endogenous or heterologous, depending on the type of yeast cell being engineered. For example, In Saccharomyces cerevisiae, the S. cerevisiae ILV5 coding region is endogenous while the Kluveromyces lactis ILV5 coding region is heterologous. Examples of KARI's that may be expressed either as endogenous or heterologous coding regions and proteins (depending on the host yeast cell) for overexpression of KARI activity in yeast mitochondria include, but are not limited to, those from ILV5 genes of Saccharomyces cerevisiae (DNA SEQ ID NO:143; protein SEQ ID NO:144), Candida glabrata (DNA SEQ ID NO151; protein SEQ ID NO:152), Kluveromyces lactis (DNA SEQ ID NO:153; protein SEQ ID NO:154), Ashbya gossypii (DNA SEQ ID NO:155; protein SEQ ID NO:156), Pichia stipitis (DNA SEQ ID NO:157; protein SEQ ID NO:158), Yarrowia lipolytica (DNA SEQ ID NO:159; protein SEQ ID NO:160), and Schizosaccharomyces pombe (DNA SEQ ID NO:161; protein SEQ ID NO:162).
In addition, bacterial KARIs may be expressed in the present yeast cells to overexpress KARI activity. When using a bacterial KARI a mitochondrial targeting signal is operably linked to the enzyme as described above for α-keto acid decarboxylase. KARI coding regions and proteins are listed in the definitions above. In addition, KARI enzymes with particularly high activities are disclosed in US Published Patent Application US20080261230. Examples of high activity KARIs disclosed therein are those from Vibrio cholerae (DNA: SEQ ID NO:15; protein SEQ ID NO:146), Pseudomonas aeruginosa PAO1, (DNA: SEQ ID NO:147; protein SEQ ID NO:148), and Pseudomonas fluorescens PF5 (DNA: SEQ ID NO:149; protein SEQ ID NO:150).
Other KARI proteins that may be used in the present yeast cells may be identified by one skilled in the art through bioinformatics methods as described above. Additional proteins that have at least about 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to SEQ ID NOs:138, 140, 142, 144, or 146 and having ketol-acid reductoisomerase activity may be used. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. In addition, experimental methods as described above for identifying α-keto acid decarboxylase coding regions and proteins may be used to identify other KARI coding regions and proteins which may be used in the present yeast cells.
A yeast ILV5 KARI coding region may be expressed using its own promoter and terminator as in Example 1 herein, or it may be part of a chimeric gene using a heterologous promoter and/or terminator. A bacterial KARI coding region is expressed in a chimeric gene using a promoter and terminator active in yeast cells. Examples of promoters, terminators, and vectors for cloning and expression of genes in yeast, as well as introduction methods, are provided above in describing α-keto acid decarboxylase expression.
An alternative method for overexpressing endogenous KARI, or other endogenous gene described below, is to replace the natural transcription control elements. For example, the natural promoter may be replaced with a promoter that is more highly expressed. Endogenous gene chromosomal promoter replacement may be performed typically using homologous recombination methods, for example as described in Mnaimneh et al. ((2004) Cell 118(1):31-44).
Overexpression of DHAD in yeast mitochondria may be achieved using endogenous or heterologous DHAD. Yeast DHAD is encoded by an ILV3 gene. ILV3 encodes DHAD and includes a mitochondrial targeting signal for translocation of the protein to the mitochondria. Bacterial DHADs which may be used do not include a mitochondrial targeting signal. To express mitochondrial activity, either a mitochondrial targeting signal is operably linked to the enzyme or a gene comprising the bacterial DHAD coding region is transformed into the mitochondrial genome, as described above for α-keto acid decarboxylase expression.
Coding sequences for DHADs that may be used herein may be derived from bacterial, fungal, or plant sources. DHADs that may be used may have a [4Fe-4S] 2+ cluster or a [2Fe-2S] 2+ cluster bound by the apoprotein. Tables 3, 4, and 5 list SEQ ID NOs for coding regions and proteins of representative DHADs that may be used in the present invention. Proteins with at least about 95% identity to those listed sequences have been omitted for simplification, but it is understood that the omitted proteins with at least about 95% sequence identity to any of the proteins listed in Tables 3, 4, and 5 and having DHAD activity may be used as disclosed herein. Additional DHAD proteins and their encoding sequences may be identified by BLAST searching of public databases, as well known to one skilled in the art. Typically BLAST (described above) searching of publicly available databases with known DHAD sequences, such as those provided herein, is used to identify DHADs and their encoding sequences that may be expressed in the present cells. For example, DHAD proteins having amino acid sequence identities of at least about 80-85%, 85%-90%, 90%-95% or 98% sequence identity to any of the DHAD proteins of Tables 3, 4, and 5 may be expressed in the present yeast cells. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix over the full length of the protein sequence.
Additional [2Fe-2S] DHADs may be identified using the analysis described in U.S. Patent Application No. 61/100,792, which is herein incorporated by reference. Therein a Profile Hidden Markov Model (HMM) was prepared based on amino acid sequences of eight functionally verified DHADs. These DHADs are from Nitrosomonas europaea (DNA SEQ ID NO:471; protein SEQ ID NO:472), Synechocystis sp. PCC6803 (DNA SEQ ID:459; protein SEQ ID NO:460), Streptococcus mutans (DNA SEQ ID NO:329; protein SEQ ID NO:330), Streptococcus thermophilus (DNA SEQ ID NO:325; SEQ ID NO:326), Ralstonia metallidurans (DNA SEQ ID NO:507; protein SEQ ID NO:508), Ralstonia eutropha (DNA SEQ ID NO:343; protein SEQ ID NO:344), and Lactococcus lactis (DNA SEQ ID NO:505; protein SEQ ID NO:506). In addition the DHAD from Flavobacterium johnsoniae (DNA SEQ ID NO:391; protein SEQ ID NO:392) was found to have dihydroxy-acid dehydratase activity when expressed in E. coli and was used in making the Profile. The Profile HMM is prepared using the HMMER software package (The theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531), following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, Va.). The output of the HMMER software program is a Profile Hidden Markov Model (HMM) that characterizes the input sequences. The Profile HMM prepared for the eight DHAD proteins is given in Table 7. Any protein that matches the Profile HMM with an E value of <10−5 is a DHAD related protein, which includes [4Fe-4S] DHADs, [2Fe-2S] DHADs, aldonic acid dehydratases, and phosphogluconate dehydratases. Sequences matching the Profile HMM are then analyzed for the presence of the three conserved cysteines, corresponding to positions 56, 129, and 201 in the Streptococcus mutans DHAD. The presence of all three conserved cysteines is characteristic of proteins having a [2Fe-2S] cluster. Proteins having the three conserved cysteines include arabonate dehydratases and [2Fe-2S] DHADs. The [2Fe-2S] DHADs may be distinguished from the aldonic acid dehydratases by analyzing for signature conserved amino acids found to be present in the [2Fe-2S] DHADs or in the aldonic acid dehydratases at positions corresponding to the following positions in the Streptococcus mutans DHAD amino acid sequence. These signature amino acids are in [2Fe-2S] DHADs or in aldonic acid dehydratases, respectively, at the following positions (with greater than 90% occurance): 88 asparagine vs glutamic acid; 113 not conserved vs glutamic acid; 142 arginine or asparagine vs not conserved; 165: not conserved vs glycine; 208 asparagine vs not conserved; 454 leucine vs not conserved; 477 phenylalanine or tyrosine vs not conserved; and 487 glycine vs not conserved.
Additionally, the sequences of DHAD coding regions provided herein may be used to identify other homologs experimentally as described above for α-keto acid decarboxylase.
Examples of promoters, terminators, and vectors for cloning and expression of genes in yeast, as well as introduction methods, are provided above in describing α-keto acid decarboxylase expression.
Acteolactate synthase (ALS) activity is also in the pathway for biosynthesis, from pyruvate, of the α-ketoisovalerate substrate of the introduced mitochondrial α-keto acid decarboxylase activity disclosed above. Though applicants found that overexpression in S. cerevisiae of the endogenous ILV2 gene encoding acetolactate synthase did not improve isobutanol production in the present yeast cells, expression of an alternate ALS in the yeast mitochondria may increase isobutanol production. Thus the present yeast cells may be engineered to express a heterologous ALS enzyme in the mitochondria. An ALS from a heterologous yeast may be expressed, which includes a mitochondrial targeting signal sequence. Examples that are heterologous when expressed in a yeast that is not the host of sequence origin include ALS from Saccharomyces cerevisiae (DNA SEQ ID NO:727; protein SEQ ID NO:728), Candida glabrata (DNA SEQ ID NO:707; protein SEQ ID NO:708), Kluveromyces lactis (DNA SEQ ID NO:709; protein SEQ ID NO:710), Ashbya gossypii (DNA SEQ ID NO:711; protein SEQ ID NO:712), Pichia stipitis (DNA SEQ ID NO:713; protein SEQ ID NO:714), Yarrowia lipolytica (DNA SEQ ID NO:715; protein SEQ ID NO:716), and Aspergillus nidulans (DNA SEQ ID NO:717; protein SEQ ID NO:718).
A bacteria ALS may be expressed as a heterologous ALS. To express mitochondrial ALS activity, either a mitochondrial targeting signal is operably linked to the enzyme or a gene comprising the bacterial ALS coding region is transformed into the mitochondrial genome, as described above for α-keto acid decarboxylase expression. Examples of bacterial ALS that may be used in the present yeast cells include those from Bacillus subtilis (DNA: SEQ ID NO:689; protein: SEQ ID NO:690), Klebsiella pneumoniae (DNA: SEQ ID NO:691; protein: SEQ ID NO:692), Lactococcus lactis (DNA: SEQ ID NO:693; protein: SEQ ID NO:694), Staphylococcus aureus, (DNA: SEQ ID NO:695; protein: SEQ ID NO:696), Listeria monocytogenes (DNA: SEQ ID NO:697; protein: SEQ ID NO:10 698), Streptococcus mutans (DNA: SEQ ID NO:699; protein: SEQ ID NO:700), Streptococcus thermophilus (DNA: SEQ ID NO:701; protein: SEQ ID NO:702), Vibrio angustum (DNA: SEQ ID NO:703; protein: SEQ ID NO:704), and Bacillus cereus (DNA: SEQ ID NO:705; protein: SEQ ID NO:706). Additional ALS coding regions and proteins may be identified using these sequences in bioinformatics or experimental methods as described above. ALS proteins with at least about 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to the SEQ ID NOs above and having acetolactate synthase activity may be used. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
Examples of promoters, terminators, and vectors for cloning and expression of genes in yeast, as well as introduction methods, are provided above in describing α-keto acid decarboxylase expression.
Over-Expressed Mitochondrial NADH Kinase
In the present yeast cells a mitochondrial NADH kinase may be over-expressed to improve isobutanol production. NADPH is required for activity of the KARI enzyme (see FIG. 1) which is in the pathway for production of the α-ketoisovalerate substrate of the introduced mitochondrial α-keto acid decarboxylase activity disclosed above. Increased mitochondrial NADH kinase activity provides increased NADPH availability to support increased KARI activity and increased α-ketoisovalerate substrate for the introduced mitochondrial α-keto acid decarboxylase.
In S. cerevisiae an NADH kinase that is localized to the mitochondrial matrix is encoded by the POS5 gene. Mitochondrial NADH kinase may be overexpressed by introducing a gene comprising the coding region of the POS5 gene or by replacing regulatory regions of the PO5 gene with higher expression regulatory regions as described above. An introduced gene comprising the coding region of the POS5 gene may include the natural regulatory regions or heterologous regulatory regions as described above. Examples of promoters, terminators, and vectors for cloning and expression of genes in yeast, as well as introduction methods, are provided above in describing α-keto acid decarboxylase expression.
Any coding region and protein with mitochondrial NADH kinase activity may be expressed in the present yeast cells. Examples include, but are not limited to, POS5 from S. cerevisiae (DNA SEQ ID NO:719; protein SEQ ID NO:720), Candida glabrata (DNA SEQ ID NO:721; protein SEQ ID NO:722), Kluyveromyces lactis (DNA SEQ ID NO:723; protein SEQ ID NO:724), and Pichia stipitis (DNA SEQ ID NO:725; protein SEQ ID NO:726). Other NADH kinase proteins that may be used in the present yeast cells may be identified by one skilled in the art through bioinformatics methods as described above. Additional proteins that have at least about 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to SEQ ID NOsisted above, and having NADH kinase activity may be used. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. In addition, experimental methods as described above for identifying α-keto acid decarboxylase coding regions and proteins may be used to identify other NADH kinase coding regions and proteins which may be used in the present yeast cells.
Any coding regions expressed in the present yeast cells may be codon optimized for expression in the specific host yeast cell being engineered as is well known to one skilled in the art. For example, for expression of the K. lactis and P. stipitis ILV3 coding regions in S. cerevisiae, each was codon optimized for S. cerevisiae expression in Example 1 herein.
Yeast Cells with Reduced Competing Pathways for Mitochondrial Osobutanol Production
Applicants have found that combining the engineering of yeast mitochondria for expression of genes as disclosed above with reducing expression of genes involved in mitochondrial branched chain amino acid biosynthesis and pyruvate metabolism gives improvement in isobutanol production.
In one embodiment the present yeast cells have one or more of these reductions in branched chain amino acid biosynthesis and pyruvate metabolism enzymes, as well as introduced mitochondrial α-keto acid decarboxylase activity, overexpressed ketol-acid reductoisomerase activity in the mitochondria, and overexpressed dihydroxy-acid dehydratase activity in the mitochondria as described above. Specifically, the present yeast cells may have reduced activity of threonine deaminase and reduced activity of isopropylmalate synthase in the mitochondria. Additionally the present yeast cells may have reduced pyruvate dehydrogenase activity in the mitochondria, and optionally reduced activity of branched chain amino acid transaminase in the mitochondria.
Eliminating threonine deaminase activity may affect pathway intermediate flow in the pathway from threonine to isoleucine. Eliminating isopropylmalate synthase activity may reduce metabolism of the α-ketoisovalerate intermediate in the leucine pathway. Eliminating mitochondrial branched chain amino acid aminotransferase activity may reduce metabolism of the alpha-ketoisovalerate intermediate in the valine pathway that is fully within the mitochondrion.
In the yeast mitochondria pyruvate is also converted to acetyl-CoA through pyruvate dehydrogenase activity (see FIG. 1). Applicants found that eliminating pyruvate dehydrogenase activity in the mitochondria further increased isobutanol production, suggesting that flow of pyruvate to alpha-ketoisovalerate was increased. The conversion of pyruvate to acetyl-CoA is catalyzed by a multienzyme pyruvate dehydrogenase complex. The pyruvate dehydrogenase enzyme is one enzyme of the multienzyme pyruvate dehydrogenase complex. Pyruvate dehydrogenase (EC 1.2.4.1) itself has alpha and beta subunits: PDA1 and PDB1, respectively, forming the E1α and E1β subunits, respectively, of the E1 component. The complex includes an E2 core which has dihydrolipoamide acetyltransferase activity (EC 2.3.1.12) and E3 which has dihydrolipoamide dehydrogenase activity (EC1.8.1.4). E2 may be encoded by LAT1 and E3 by LPD1. An additional complex protein is encoded by PDX1, which links Lat1p to Lpd1p. Thus the pyruvate dehydrogenase complex may include PDA1, PDB1, Lat1, Lpd1, and Pdx1, or homologous proteins encoded by genes which may have alternative names in various yeasts. The activity of any of these proteins may be reduced to affect the function of the pyruvate dehydrogenase complex, and thereby affect pyruvate dehydrogenase activity, to prepare a strain of one embodiment of the present invention. In the description below when referring to PDA1, it is understood that PDA1 may be substituted by any of PDB1, LAT1, LPD1, or PDX1, any of which may be modified to reduce pyruvate dehydrogenase activity.
In the present invention, any yeast enzymes providing threonine deaminase, isopropylmalate synthase, branched chain amino acid aminotransferase, or pyruvate dehydrogenase activities in the mitochondria may be targets for engineering to reduce these activities. Preferably the activity is reduced such that there is substantially no detectable activity of the target enzyme. Yeast cells are engineered to reduce enzyme activity typically by modification of the gene encoding the target enzyme. The genes encoding these enzymes are ILV1, LEU4, BAT1, and PDA1 (and miltienzyme complex genes PDB1, LAT1, LPD1, and PDX1), respectively. Any ILV1, LEU4, BAT1, or PDA1 gene of yeast encoding a mitochondrial targeted protein is a target for engineering for reduced expression of the encoded enzyme activity in the present cells. Examples of target coding region sequences and their encoded proteins from different species of yeast cells are given in Table 1. Other target proteins, or their encoding sequences, having at least about 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95%, or at least about 98% sequence identity to any of the proteins or coding sequences listed in Table 1, and having one of these activities, may be identified in the literature and in bioinformatics databases well known to the skilled person.
There is cytoplasmic isopropylmalate synthase activity encoded by LEU9 and cytoplasmic branched chain amino acid transaminase activity encoded by BAT2, genes which are not targets in the present disclosure.
Because mitochondrial threonine deaminase, isopropylmalate synthase, branched chain amino acid aminotransferase, and pyruvate dehydrogenase complex enzymes are well known, as well as their encoding genes (ILV1, LEU4, BAT1, PDA1, PDB1, LAT1, LPD1, and PDX1, respectively), one skilled in the art can readily identify these proteins and their encoding genes in yeast cells using bioinformatics approaches, to identify additional target genes for engineering as disclosed herein. Typically BLAST (described above) searching of publicly available databases with known target protein sequences, such as those provided herein, is used to identify homologous proteins and their encoding sequences that may be targeted for inactivation in the present strains. For example, endogenous yeast mitochondrial threonine deaminase proteins having amino acid sequence identities of at least about 80-85%, 85%-90%, 90%-95% or 98% sequence identity to any of the threonine deaminase proteins of SEQ ID NOs:2, 3, 5, 7, 9, 11, or 13 may have reduced expression in the present strains. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
In the following description, ILV1 is used as an example, and the same description applies to any of LEU4, BAT1, PDA1, PDB1, LAT1, LPD1, and PDX1 coding regions. The sequences of, for example, the ILV1 coding regions provided herein may be used to identify other homologs in nature. For example each of the threonine deaminase encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.
For example, genes encoding similar proteins or polypeptides to the threonine deaminase encoding genes provided herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired organism using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the disclosed nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments by hybridization under conditions of appropriate stringency.
Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.). Further description is presented above, exemplified for α-keto acid decarboxylases.
Threonine deaminase and isopropylmalate synthase, and optionally branched chain amino acid aminotransferase and/or pyruvate dehydrogenase activities may be reduced using genetic manipulations that disrupt expression of active enzyme from the target gene. Many methods for genetic modification of target genes are known to one skilled in the art and may be used to create the present yeast strains. Modifications that may be used to reduce or eliminate expression of a target protein are disruptions that include, but are not limited to, deletion of the entire gene or a portion of the gene, inserting a DNA fragment into the gene (in either the promoter or coding region) so that the protein is not expressed or expressed at lower levels, introducing a mutation into the coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the coding region to alter amino acids so that a non-functional or a less enzymatically active protein is expressed. In addition, expression of a gene may be blocked by expression of an antisense RNA or an interfering RNA, and constructs may be introduced that result in cosuppression. In addition, the synthesis or stability of the transcript may be lessened by mutation. Similarly the efficiency by which a protein is translated from mRNA may be modulated by mutation. In addition, since the target proteins are all mitochondrial, disruption of mitochondrial localization may be used such as disrupting the mitochondrial targeting signal sequence. All of these methods may be readily practiced by one skilled in the art making use of the known or identified coding sequences as exemplified in Table 1.
DNA sequences surrounding a target gene coding sequence are also useful in some modification procedures and are available for yeasts such as for Saccharomycse cerevisiae in the complete genome sequence coordinated by Genome Project ID9518 of Genome Projects coordinated by NCBI (National Center for Biotechnology Information) with identifying GOPID #13838. Additional examples of yeast genomic sequences include that of Yarrowia lipolytica, GOPIC #13837, and of Candida albicans, which is included in GPID #10771, #10701 and #16373. Additional genomes have been completely sequenced and annotated and are publicly available for the following yeast strains Candida glabrata CBS 138, Kluyveromyces lactis NRRL Y-1140, Pichia stipitis CBS 6054, and Schizosaccharomyces pombe 972h-.
In particular, DNA sequences surrounding a target coding sequence are useful for modification methods using homologous recombination. For example, in this method flanking sequences are placed bounding a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the target gene. Also partial target gene sequences and flanking sequences bounding a selectable marker gene may be used to mediate homologous recombination whereby the marker gene replaces a portion of the target gene. In addition, the selectable marker may be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the target gene without reactivating the latter. The site-specific recombination leaves behind a recombination site which disrupts expression of the target gene encoded protein. The homologous recombination vector may be constructed to also leave a deletion in the target gene following excision of the selectable marker, as is well known to one skilled in the art.
Deletions may be made using mitotic recombination as described in Wach et al. ((1994) Yeast 10:1793-1808). This method involves preparing a DNA fragment that contains a selectable marker between genomic regions that may be as short as 20 bp, and which bound a target DNA sequence. This DNA fragment can be prepared by PCR amplification of the selectable marker gene using as primers oligonucleotides that hybridize to the ends of the marker gene and that include the genomic regions that can recombine with the yeast genome. The linear DNA fragment can be efficiently transformed into yeast and recombined into the genome resulting in gene replacement including with deletion of the target DNA sequence (as described in Methods in Enzymology, v194, pp 281-301 (1991)).
Moreover, promoter replacement methods may be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression such as described in Mnaimneh et al. ((2004) Cell 118(1):31-44) and in Example 12 herein.
In addition, a target gene in any yeast cell may be disrupted using random mutagenesis, which is followed by screening to identify strains with reduced target gene encoded activity. Using this type of method, the DNA sequence of any region of the genome affecting expression of a target protein need not be known. Methods for creating genetic mutations are common and well known in the art and may be applied to the exercise of creating mutants. Commonly used random genetic modification methods (reviewed in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) include spontaneous mutagenesis, mutagenesis caused by mutator genes, chemical mutagenesis, irradiation with UV or X-rays, or transposon mutagenesis.
Chemical mutagenesis of yeast commonly involves treatment of yeast cells with one of the following DNA mutagens: ethyl methanesulfonate (EMS), nitrous acid, diethyl sulfate, or N-methyl-N′-nitro-N-nitroso-guanidine (MNNG). These methods of mutagenesis have been reviewed in Spencer et al (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). Chemical mutagenesis with EMS may be performed as described in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Irradiation with ultraviolet (UV) light or X-rays can also be used to produce random mutagenesis in yeast cells. The primary effect of mutagenesis by UV irradiation is the formation of pyrimidine dimers which disrupt the fidelity of DNA replication. Protocols for UV-mutagenesis of yeast can be found in Spencer et al (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). Introduction of a mutator phenotype can also be used to generate random chromosomal mutations in yeast. Common mutator phenotypes can be obtained through disruption of one or more of the following genes: PMS1, MAG1, RAD18 or RAD51. Restoration of the non-mutator phenotype can be easily obtained by insertion of the wildtype allele. Collections of modified cells produced from any of these or other known random mutagenesis processes may be screened for reduced activity of the target enzyme.
Production of Isobutanol
Strains of yeast disclosed herein may be grown in fermentation media for production of isobutanol. Carbon substrates suitable for isobutanol production may include but are not limited to monosaccharides such as fructose, oligosaccharides such as lactose maltose, galactose, or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include ethanol, lactate, succinate, or glycerol.
Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates.
Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars. Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars may be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Application Publication No. 2007/0031918A1, which is herein incorporated by reference. Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
In addition to an appropriate carbon source, fermentation media typically contains suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for production of the desired product.
By-Product Formation
It will be appreciated that reduction and preferably elimination of by-products of carbon metabolism other than carbon dioxide and isobutanol would be advantageous for production of isobutanol. For example microorganisms metabolizing sugar substrates produce a variety of by-products in a mixed acid fermentation (Moat, A. G. et al., Microbial Physiology, 4th edition, John Wiley Publishers, N.Y., 2002). Yeast metabolizing sugar substrates produce a variety of by-products like acids and alcohols such as, but not limited to, formate, lactate, succinate, ethanol, acetate and glycerol. Formation of these byproducts during isobutanol fermentation lower the yield of isobutanol. To prevent yield loss of isobutanol the genes encoding enzyme activities corresponding to byproduct formation can be down-regulated or disrupted using methods described herein and/or known in the art.
Endogenous pyruvate decarboxylase activity in yeast converts pyruvate to acetaldehyde, which is then converted to ethanol or to acetyl-CoA via acetate. Therefore, endogenous pyruvate decarboxylase activity is a target for reduction of byproduct formation. Yeasts may have one or more genes encoding pyruvate decarboylase. For example, there is one gene encoding pyruvate decarboxylase in Kluyveromyces lactis, while there are three isozymes of pyruvate decarboxylase encoded by the PDC1, PCD5, and PDC6 genes in Saccharomyces cerevisiae, as well as a pyruvate decarboxylase regulatory gene PDC2. Expression of pyruvate decarboxylase from PDC6 is minimal. In yeast strains disclosed herein, the pyruvate decarboxylase activity may be reduced by downregulating or disrupting at least one gene encoding a pyruvate decarboxylase, or a gene regulating pyruvate decarboxylase gene expression as described in U.S. patent application Ser. No. 12/477,942, which is herein incorporated by reference. For example, in S. cerevisiae the PDC1 and PDC5 genes, or all three genes, may be disrupted. Alternatively, pyruvate decarboxylase activity may be reduced by disrupting the PDC2 regulatory gene in S. cerevisiae. In other yeasts, genes encoding pyruvate decarboxylase proteins such as those having at least about 80-85%, 85%-90%, 90%-95%, or at least about 98% sequence identity to PDC1 or PDC5 may be downregulated or disrupted. Examples of yeast pyruvate decarboxylase genes or proteins that may be targeted for downregulation or disruption are listed in Table 14 (SEQ ID NOs: 728, 730, 732, 734, 736, 738, 740, 742, and 744).
Examples of yeast strains with reduced pyruvate decarboxylase activity due to disruption of pyruvate decarboxylase encoding genes have been reported such as for Saccharomyces in Flikweert et al. (Yeast (1996) 12:247-257), for Kluyveromyces in Bianchi et al. (Mol. Microbiol. (1996) 19(1):27-36), and disruption of the regulatory gene in Hohmann, (Mol Gen Genet. (1993) 241:657-666). Saccharomyces strains having no pyruvate decarboxylase activity are available from the ATCC (Accession #200027 and #200028).
Culture Conditions
Typically cells are grown at a temperature in the range of about 20° C. to about 37° C. in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as broth that includes yeast nitrogen base, ammonium sulfate, and dextrose as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.
Suitable pH ranges for the fermentation are between pH 3.0 to pH 7.5, where pH 4.5.0 to pH 6.5 is preferred as the initial condition.
Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.
The amount of isobutanol produced in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).
Industrial Batch and Continuous Fermentations
The present process employs a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.
A variation on the standard batch system is the Fed-Batch system. Fed-Batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.
Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
It is contemplated that the present invention may be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isobutanol production.
Methods for Isobutanol Isolation from the Fermentation Medium
Bioproduced isobutanol may be isolated from the fermentation medium using methods known in the art. For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the isobutanol may be isolated from the fermentation medium, which has been treated to remove solids as described above, using methods such as distillation, liquid-liquid extraction, or membrane-based separation. Because isobutanol forms a low boiling point, azeotropic mixture with water, distillation can only be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify isobutanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, isobutanol may be isolated using azeotropic distillation using an entrainer (see for example Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).
The isobutanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the isobutanol. In this method, the isobutanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the isobutanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The isobutanol-rich decanted organic phase may be further purified by distillation in a second distillation column.
The isobutanol may also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the isobutanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The isobutanol-containing organic phase is then distilled to separate the isobutanol from the solvent.
Distillation in combination with adsorption may also be used to isolate isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).
Additionally, distillation in combination with pervaporation may be used to isolate and purify the isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199-210 (2004)).
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
General Methods
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987), and by Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following Examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified. Microbial strains were obtained from The American Type Culture Collection (ATCC), Manassas, Va., unless otherwise noted. The oligonucleotide primers used in the following Examples are given in Table 3. All the oligonucleotide primers were synthesized by Sigma-Genosys (Woodlands, Tex.) or Integrated DNA Technologies (Coralsville, Iowa).
Synthetic complete medium is described in Amberg, Burke and Strathern, 2005, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
GC Method
The GC method utilized an HP-InnoWax column (30 m×0.32 mm ID, 0.25 μm film) from Agilent Technologies (Santa Clara, Calif.). The carrier gas was helium at a flow rate of 1 ml/min measured at 150° C. with constant head pressure; injector split was 1:10 at 200° C.; oven temperature was 45° C. for 1 min, 45° C. to 230° C. at 10° C./min, and 230° C. for 30 sec. FID detection was used at 260° C. with 40 ml/min helium makeup gas. Culture broth samples were filtered through 0.2 μM spin filters before injection. Depending on analytical sensitivity desired, either 0.1 μl or 0.5 μl injection volumes were used. Calibrated standard curves were generated for the following compounds: ethanol, isobutanol, acetoin, meso-2,3-butanediol, and (2S,3S)-2,3-butanediol. Analytical standards were also utilized to identify retention times for isobutryaldehyde, isobutyric acid, and isoamyl alcohol.
The meaning of abbreviations is as follows: “s” means second(s), “min” means minute(s), “h” means hour(s), “psi” means pounds per square inch, “nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” means nanometers, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmol” means micromole(s)”, “g” means gram(s), “μg” means microgram(s) and “ng” means nanogram(s), “PCR” means polymerase chain reaction, “OD” means optical density, “OD600” means the optical density measured at a wavelength of 600 nm, “kDa” means kilodaltons, “g” means the gravitation constant, “bp” means base pair(s), “kbp” means kilobase pair(s), “% w/v” means weight/volume percent, % v/v” means volume/volume percent, “wt %” means percent by weight, “HPLC” means high performance liquid chromatography, “GC” means gas chromatography, “FID” means flame ionization detector.
Example 1
Vector Construction for KARI and DHAD Expression
The purpose of this example is to demonstrate how to construct yeast expression vectors for expression of ketol acid reductoisomerase (KARI) and/or dihydroxy isovalerate dehydratase (DHAD) in the yeast mitochondria. KARI is encoded by the ILV5 gene and DHAD is encoded by the ILV3 gene. Both KARI and DHAD coding regions from yeast include sequences encoding mitochondrial targeting signals for localization of these proteins in the mitochondria.
Creation of DHAD Constructs:
pRS423::FBAp-ILV3(KL)-M(O).
To express the ILV3 gene from Kluyveromyces lactis, the coding region was codon-optimized for expression in S. cerevisiae, and a DNA fragment of this sequence was synthesized and cloned by DNA 2.0 (Menlo Park, Calif.). The synthesized ILV3 coding region fragment (SEQ ID NO:118) was isolated and cloned into the vector pRS423::FBAp (SEQ ID NO:80) with SphI and NotI. The pRS423::FBAp vector is also called pNY13. The pNY13 shuttle vector contained an F1 origin of replication (1423 to 1879) for maintenance in E. coli and a 2 micron origin (nt 7537 to 8881) for replication in yeast. The vector has an FBA promoter (nt 2111 to 3110) and FBA terminator (nt 4316 to 5315). In addition, it carries the HIS3 marker (nt 504 to 1163) for selection in yeast and ampicillin resistance marker (nt 6547 to 7404) for selection in E. coli. The resulting construct was named pRS423::FBAp-ILV3(KL).
pRS423::FBAp-ILV3(PS)-M(O).
To express the ILV3 gene from Pichia stipitis, the coding region was codon-optimized for expression in S. cerevisiae and a DNA fragment of this sequence was synthesized and cloned by DNA 2.0. The synthesized ILV3 coding region fragment was cloned into the same vector pRS423::FBAp (pNY13) described above with SphI and NotI. The resulting construct was named pRS423::FBAp-ILV3(PS)-M(O).
Creation of DHAD/KARI Constructs:
pRS423::FBAp-ILV3(KL)+nILV5
To combine the expression of ILV5 and ILV3 from K. lactis on a single vector, the native ILV5 gene from S. cerevisiae, including the promoter, coding region and terminator, was amplified from BY4741 (ATCC #201388) genomic DNA using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers ILV5-R(Xho) (SEQ ID NO:81) and ILV5-F(XhoPstAsc) (SEQ ID NO:82). The PCR product was digested with XhoI and AscI and ligated into the corresponding sites in the plasmid pRS423::FBAp-ILV3(KL)-M(O). The resulting construct was named pRS423 FBAp-ILV3(KL)+nILV5.
pRS423::FBAp-ILV3(PS)+nILV5
To combine the expression of ILV5 and ILV3 from P. stipitis on a single vector, the native ILV5 gene from S. cerevisiae, including the promoter, coding region and terminator, was amplified from BY4741 (ATCC #201388) genomic DNA using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-5405) and primers ILV5-R(Xho) (SEQ ID NO:81) and ILV5-F(XhoPstAsc) (SEQ ID NO:82). The PCR product was digested with XhoI and AscI and ligated into the corresponding sites in plasmid pRS423::FBAp-ILV3(PS)-M(O). The resulting construct was named pRS423 FBAp-ILV3(PS)+nILV5.
Example 2
Vector Construction for Expression of Alpha-Ketoisovalerate Decarboxylase and Acetolactate Synthase
The purpose of this example is to demonstrate how to construct yeast expression vectors that express alpha-ketoisovalerate decarboxylase (KivD) and acetolactate synthase (ALS; ILV2 gene) in the yeast mitochondria. The ILV2 coding region from yeast includes a sequence encoding a mitochondrial targeting signal for localization of the protein in the mitochondria.
Creation of Mitochondria-Targeted KivD Constructs:
pRS426::GPM-kivD: Plasmid pRS426::FBA-ILV5-GPM-kivD was described in US Patent Publication US20070092957 A1, Example 17 which is herein incorporated by reference. Plasmid pRS426::FBA-ILV5-GPM-kivD is vector pRS426 (ATCC #77107) containing a chimeric gene including an FBA promoter, ILV5 coding region and CYC terminator, and a second chimeric gene containing a GPM promoter (SEQ ID NO:113), kivD coding region from Lactococcus lactis that was codon optimized for expression in E. coli (SEQ ID NO:114), and ADH1 terminator (SEQ ID NO:115). pRS426::FBA-ILV5-GPM-kivD was digested with SacII and NotI to remove the FBA-ILV5-CYC terminator gene. The remaining vector backbone was treated with T4 DNA polymerase to generate blunt ends, was ligated to re-form a circularized product, and was transformed into E. coli Top10 cells. The resulting plasmid was named pRS426::GPM-kivD (SEQ ID NO:83).
pRS426::GPM-MTSkivD
In order to target the Lactococcus lactis KivD enzyme to the yeast mitochondria, the 25 amino acid mitochondrial targeting sequence from cytochrome C oxidase (COX) subunit IV of S. cerevisiae (DNA SEQ ID NO:116, protein SEQ ID NO:117; Hurt et al. EMBO J. (1984) 3(13):3149-56) was added to the N-terminus of KivD. This COX mitochondrial targeting sequence was added by PCR-amplification of kivD from pRS426::GPM-kivD using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers 112590-80A (SEQ ID NO:84) and 112590-80D (SEQ ID NO:85). The COX mitochondrial targeting sequence was present in primer 112590-80A. The PCR product was digested with BbvCI and AgeI, and ligated into BbvCI, AgeI, and CIP-digested pRS426-GPM-kivD. This digestion removed the N-terminal portion of the original kivD coding region, and replaced it with the mitochondrial targeting sequence fused to the N-terminal kivD sequence. The mitochondrial targeting sequence in the resulting plasmid, pRS426-GPM-MTSkivD was verified by DNA sequencing using primer N133SeqR4 (SEQ ID NO:86).
Creation of Acetolactate Synthase (ALS) Constructs
The native ILV2 locus, consisting of the promoter, coding region and terminator, was PCR-amplified from BY4743 genomic DNA using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-5405) with primers 112590-91A (SEQ ID NO:87) and 112590-91B (SEQ ID NO:88), which added 5′ SacI 3′ AvrII restriction sites. The PCR product was digested with SacI and AvrII, and ligated into pRS426-GPM-MTSkivD (described above) which was also digested with SacI and SpeI, then digested with CIP. The resulting plasmid, pRS426-nILV2-GPM-MTSkivD was verified by diagnostic restriction digestion and DNA sequencing using primers 112590-91A (SEQ ID NO:87) and 112590-91B (SEQ ID NO:88).
Example 3
Production of Isobutanol in Yeast Mitochondria
The purpose of this example is to demonstrate isobutanol production in the mitochondria of S. cerevisiae using the expression plasmids above that contain ILV3, ILV5, and either kivD or MTSkivD. Note that endogenous acetolactate activity from ILV2 is used in this example, as well as endogenous alcohol dehydrogenase activity.
Plasmids pRS423 containing the native ILV5 locus and FBAp-ILV3 cassette (Pichia or Kluyveromyces) and either pRS426 containing cytosolic kivD or MTSkivD were transformed into strain BY4741 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and transformants were maintained on synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose. The strains were grown in synthetic complete media lacking histidine and uracil and supplemented with 2% glucose under aerobic conditions (20 ml media in 125 ml flask) and cultured at 30° C. shaking at 220 rpm. Cultures were inoculated at 0.1 OD600 and assayed for isobutanol titers at 48 hours post-inoculation. Isobutanol was quantitated by GC-FID on a HP-Innowax column using a standard curve of pure isobutanol and results are given in Table 9.







TABLE 9







Isobutanol production using cytoplasmic or mitochondrial KivD











Isobutanol (mM)



Strain
48 hours







BY4741 pRS423/pRS426
0.07



BY4741 pRS423-nILV5-FBAp-
0.18



ILV3(KL)/pRS426



BY4741 pRS423-nILV5-FBAp-
0.22



ILV3(KL)/pRS426-GPMp-kivD



BY4741 pRS423-nILV5-FBAp-
0.53



ILV3(KL)/pRS426-GPMp-



MTSkivD



BY4741 pRS423-nILV5-FBAp-
0.18



ILV3(PS)/pRS426



BY4741 pRS423-nILV5-FBAp-
0.20



ILV3(PS)/pRS426-GPMp-kivD



BY4741 pRS423-nILV5-FBAp-
0.48



ILV3(PS) pRS426-GPMp-



MTSkivD










Expression of ILV5 and ILV3 in the yeast mitochondria increased the basal levels of isobutanol approximately 2-fold from the parent BY4741 strain. Expression of cytosolic KivD only marginally increased isobutanol titers. However, expression of mitochondrial-targeted KivD (MTSkivD) resulted in significant increases in isobutanol production, with only a small difference between Pichia and Kluyveromyces ILV3 enzymes.

Example 4
Production of Isobutanol in Yeast Mitochondria with Endogenous ALS Overexpression
The purpose of this example is to demonstrate isobutanol production in the mitochondria of S. cerevisiae using the expression plasmids above that contain ILV3, ILV5, MTSkivD, and ILV2.
Plasmids pRS423-nILV5-FBAp-ILV3(KL) and pRS426-nILV2-GPMp-MTSkivD or pRS423-nILV5-FBAp-ILV3(PS) and pRS426-nILV2-GPMp-MTSkivD (plasmids described above) were transformed into strain BY4741 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and transformants were maintained on synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose. Three to five colonies from each strain were grown in synthetic complete media lacking amino acid dropout mix but supplemented with methionine and leucine under aerobic conditions (20 ml media in 125 ml flask) and cultured at 30° C. shaking at 220 rpm. Cultures were inoculated at 0.1 OD600 and assayed for isobutanol titers at 24 and 48 hours post-inoculation. Isobutanol was quantitated by GC-FID on a HP-Innowax column using a standard curve of pure isobutanol and results are given Table 10.







TABLE 10







Mitochondrial isobutanol production with overexpression of


endogenous ALS













Isobutanol




Isobutanol (mM)
(mM)



Strain
24 hours
48 hours







BY4741 pRS423-nILV5-
0.171 ± 0.14
0.375 ± 0.08



FBAp-ILV3(KL)/pRS426-



nILV2-GPMp-MTSkivD



BY4741 pRS423-nILV5-
0.334 ± 0.04
0.385 ± 0.01



FBAp-ILV3(PS)/pRS426-



nILV2-GPMp-MTSkivD










In this experiment addition of additional copies of the endogenous ALS gene (ILV2) did not improve isobutanol production.

Example 5
Eliminating Expression of Enzymes in Branched Chain Amino Acid Biosynthesis Pathways in S. cerevisiae 
Yeast cells were engineered to eliminate activities of specific enzymes of mitochondrial branched chain amino acid biosynthesis. Three chromosomal disruptions were generated in successive fashion in the following genes: ILV1, encoding threonine deaminase; LEU4, encoding 2-isopropylmalate synthase; and BAT1, encoding branched chain amino acid aminotransferase.
An ilv1::LEU2 cassette was constructed by PCR amplification of the LEU2 marker from pRS425 (ATCC No. 77106) using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) with primers 112590-88A (SEQ ID NO:89) and 112590-88B (SEQ ID NO:90). The ILV1 portion of each primer was derived from the 5′ region upstream of the ILV1 promoter and 3′ region downstream of the transcriptional terminator, respectively, such that integration of the LEU2 marker results in replacement of the ILV1 coding region. The  ̃1.7 kb PCR product was transformed into Saccharomyces cerevisiae strain BY4741 (ATCC #201388) with selection on synthetic complete media lacking leucine and supplemented with 2% glucose at 30° C. Transformants were screened by colony PCR using primers 112590-88C (SEQ ID NO:91) and 112590-88D (SEQ ID NO:92). The resulting identified strain had the genotype: BY4741 ilv1::LEU2.
A leu4::URA3r disruption cassette was constructed by PCR amplification of the URA3r marker from pUC19-URA3r (SEQ ID NO:93) using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-5405) and primers 112590-97A (SEQ ID NO:94) and 112590-97B (SEQ ID NO:95). pUC19-URA3r contains the URA3 marker from pRS426 (ATCC no. 77107) flanked by 75 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. The LEU4 portion of each primer was derived from the 5′ region upstream of the LEU4 promoter and 3′ region downstream of the transcriptional terminator, respectively, such that integration of the URA3r marker results in replacement of the LEU4 coding region. The  ̃1.5 kb PCR product was transformed into BY4741 ilv1::LEU2 cells with selection on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR using external primers 112590-49E (SEQ ID NO:96) and 112590-97C (SEQ ID NO:97) to verify integration at the correct site. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain had the genotype: BY4741 Δilv1 Δleu4.
A bat1::URA3r disruption cassette was constructed in several steps. A cassette containing the BAT1 5′ region was amplified from BY4741 genomic DNA using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-5405) and primers 112590-108A (SEQ ID NO:98) and 112590-108B (SEQ ID NO:99). The cassette containing BAT1 3′ sequences was amplified from BY4741 genomic DNA using Phusion DNA polymerase and primers 112590-108C (SEQ ID NO:100) and 112590-108D (SEQ ID NO:101). The URA3r marker was PCR-amplified from pUC19-URA3r using Phusion DNA polymerase and primers 112590-108E (SEQ ID NO:102) and 112590-108F (SEQ ID NO:103). The three PCR products were combined in a SOE PCR reaction (Horton et al. (1989) Gene 77:61-68) and amplified using Phusion DNA polymerase and the end primers 112590-108A (SEQ ID NO:98) and 112590-108D (SEQ ID NO:101), generating the full  ̃2.8 kb BAT1::URA3r disruption cassette. The BAT1 portion of each primer was derived from the 5′ region upstream of the BAT1 promoter and 3′ region downstream of the transcriptional terminator, respectively, such that integration of the URA3r marker results in replacement of the BAT1 coding region. The cassette was transformed into BY4741 Δilv1 Δleu4 with selection on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR using external primers 112590-49E (SEQ ID NO:96) and “BAT1 check” (SEQ ID NO:104) to verify integration at the correct site. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto synthetic complete media lacking uracil supplemented with 2% glucose to verify the absence of growth. The resulting identified strain had the genotype: BY4741 Δilv1 Δleu4 Δbat1.
Example 6
Mitochondrial Isobutanol Production in Recombinant S. cerevisiae with Amino Acid Pathway Alterations Δilv1 Δleu4
The purpose of this example is to demonstrate isobutanol production in the mitochondria of S. cerevisiae in which expression of ILV1 (threonine deaminase) and LEU4 (2-isopropylmalate synthase) is disrupted.
Plasmids pRS423-nILV5-FBAp-ILV3(PS) and pRS426-nILV2-GPMp-MTSkivD, all described above, were transformed into strain BY4741 or strain BY4741 Δilv1 Δleu4 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and transformants were maintained on synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose. BY4741 Δilv1 Δleu4 was transformed with pRS423 and pRS426 as a control. Strains were grown in synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose under aerobic conditions (20 ml media in 125 ml flask) and cultured at 30° C. shaking at 220 rpm. Cultures were inoculated at 0.1 OD600 and assayed for isobutanol titers at 24 hours post-inoculation. Isobutanol was quantitated by GC-FID on a HP-Innowax column using a standard curve of pure isobutanol and results are given Table 11.







TABLE 11







Mitochondrial isobutanol production in 2-gene amino acid


pathway modified strain











Isobutanol (mM)



Strain
24 hours







BY4741 pRS423-nILV5-FBAp-ILV3(PS)/
0.334 ± 0.04*



pRS426-nILV2-GPMp-MTSkivD



BY4741 Δilv1 Δleu4 pRS423/pRS426
0.64



BY4741 Δilv1 Δleu4 pRS423-nILV5-FBAp-
0.95



ILV3(PS)/pRS426-nILV2-GPMp-MTSkivD







*data from Example 4






Example 7
Mitochondrial Isobutanol Production in Recombinant S. cerevisiae Strain BY4741 Δilv1 Δleu4 Δbat1
The purpose of this example is to demonstrate isobutanol production in the mitochondria of S. cerevisiae in which ILV1 (threonine deaminase), LEU4 (2-isopropylmalate synthase), and BAT1 (branched-chain amino acid aminotransferase) are disrupted.
Plasmids pRS423::nILV5-FBAp-ILV3(PS) and pRS426::nILV2-GPMp-MTSkivD (plasmids described above) were transformed into strain BY4741 Δilv1 Δleu4 Δbat1 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and transformants were maintained on synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose. Strains were grown in synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose under aerobic conditions (20 ml media in 125 ml flask) and cultured at 30° C. shaking at 220 rpm. Cultures were inoculated at 0.1 OD600 and assayed for isobutanol titers at 24 hours post-inoculation. Isobutanol was quantitated by GC-FID on a HP-Innowax column using a standard curve of pure isobutanol and results are given Table 12.







TABLE 12







Mitochondrial isobutanol production in 3-gene amino acid


pathway modified strain











Isobutanol (mM)



Strain
24 hours







BY4741 Δilv1 Δleu4 Δbat1
0.90



pRS423/pRS426



BY4741 Δilv1 Δleu4 Δbat1
0.91



pRS423::nILV5-FBAp-ILV3(PS)/



pRS426::nILV2-GPMp-MTSkivD



Clone #1



BY4741 Δilv1 Δleu4 Δbat1
0.53



pRS423::nILV5-FBAp-ILV3(PS)/



pRS426::nILV2-GPMp-MTSkivD



Clone #2










Though there was variability in different clones, isobutanol production was similar in the triple disruption strain with and without mitochondrial expression of isobutanol pathway genes.

Example 8
Reduction of Pyruvate Dehydrogenase Activity in S. cerevisiae 
To reduce levels of mitochondrial pyruvate dehydrogenase activity the PDA1 gene, encoding the E1α subunit of pyruvate dehydrogenase, was altered. The native promoter was replaced with the inducible GAL1 promoter through homologous recombination. The GAL1 promoter and URA3r marker were joined together by SOE PCR. The URA3r marker was PCR amplified from pUC19-URA3r using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers 112590-118A (SEQ ID NO:105) and 112590-118B (SEQ ID NO:106). The GAL1 promoter was PCR-amplified from pRS426::GAL1p-alsS (SEQ ID NO:107) using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-5405) and primers 112590-118C (SEQ ID NO:108) and 112590-118D (SEQ ID NO:109). pRS426::GAL1p-alsS contained an F1 origin of replication (nt 4976 to 5432) for maintenance in E. coli and a 2 micron origin (nt 2215 to 3560) for replication in yeast. The vector has a GAL1 promoter (nt 7702 to 8144) and CYC1 terminator (nt 5721 to 5970). In addition, it carries the URA3 marker (nt 4042 to 4845) for selection in yeast and ampicillin resistance marker (nt 1225 to 2082) for selection in E. coli. 
The two products were joined by SOE PCR using Phusion DNA polymerase, and primers 112590-118E (SEQ ID NO:110) and 112590-118F (SEQ ID NO:111). The PDA1 portion of each primer was derived from the 5′ region upstream of the PDA1 promoter and PDA1 coding sequence, respectively, such that integration of the URA3 marker results in replacement of the native PDA1 promoter with the GAL1 promoter. The PCR product was transformed into BY4741 Δilv1 Δleu4 Δbat1 with selection on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR using external primers 112590-49E (SEQ ID NO:96) and 112590-118G (SEQ ID NO:112) to verify integration at the PDA1 locus. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5FOA plates onto synthetic complete media lacking uracil supplemented with 2% glucose to verify the absence of growth. The resulting identified strain had the genotype: BY4741 Δilv1 Δleu4 Δbat1 GAL1p-PDA1.
Example 9
Mitochondrial Isobutanol Production in Recombinant S. cerevisiae Strain BY4741 Δilv1 Δleu4 Δbat1 GAL1p-PDA1
The purpose of this example is to demonstrate isobutanol production in the mitochondria of S. cerevisiae in which ILV1 (threonine deaminase), LEU4 (2-isopropylmalate synthase), and BAT1 (branched-chain amino acid aminotransferase) were disrupted, and PDA1 (pyruvate dehydrogenase) was down-regulated.
Plasmids pRS423::nILV5-FBAp-ILV3(PS) and pRS426::GPMp-MTSkivD, described above, were transformed into strain BY4741 Δilv1 Δleu4 Δbat1 GAL1p-PDA1 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and transformants were maintained on synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose. Empty plasmid pRS423 and pRS426 were similarly transformed into BY4741 Δilv1 Δleu4 Δbat1 GAL1p-PDA as a control. Strains were grown in SD (2% glucose)-HIS-URA media under aerobic conditions (20 ml media in 125 ml flask) and were cultured at 30° C. shaking at 220 rpm. Under these growth conditions, the glucose present in the media repressed the GAL1 promoter thereby reducing expression of PDA1. Cultures were inoculated at 0.1 OD600 and assayed for isobutanol titers at 24 hours post-inoculation. Isobutanol was quantitated by GC-FID on a HP-Innowax column using a standard curve of pure isobutanol and results are given Table 13.







TABLE 13







Mitochondrial isobutanol production in amino acid pathway


and pyruvate metabolism modified strain











Isobutanol (mM)



Strain
24 hours














BY4741 Δilv1 Δleu4 Δbat1
0.90



pRS423/pRS426



BY4741 Δilv1 Δleu4 Δbat1
0.95



GAL1p-PDA1 pRS423/pRS426



BY4741 Δilv1 Δleu4 Δbat1
1.1



GAL1p-PDA1 pRS423::nILV5-



FBAp-ILV3(PS)/pRS426::GPMp-



MTSkivD










When the mitochondrial isobutanol pathway plasmids were introduced into the BY4741 Δilv1 Δleu4 Δbat1 GAL1p-PDA1 strain background, isobutanol titers increased significantly.





























TABLE 8







HMM
A
C
D
E
F
G
H
I
K











Position in



m->m
m->i
m->d
i->m
i->i
d->m
d->d
b->m
m->e
L
M
N
P
Q
R
S
T
V
W
Y
alignment






−538
*
−1684


 1(M)
−233
−1296
99
1223
−1477
−1132
89
−1122
420
−1248
1757
1553
−1296
464
−24
−190
−188
−838
−1578
−985
6


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−29
−6203
−7245
−894
−1115
−701
−1378
−538
*


 2(E)
−220
−1288
232
1356
−1807
1016
−70
−1474
190
−1584
−775
132
−1298
300
−282
−183
1140
−1092
−1872
−1262
7


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*


 3(K)
−448
−1932
1558
658
−2220
−1048
40
−1983
1569
−1938
−1091
1558
−1319
450
−193
−278
−419
−1552
−2121
−1397
8


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*


 4(V)
−404
−498
−1497
−939
−588
−1810
−640
1591
914
−127
335
−962
−1866
−562
−767
−868
−357
1720
−1169
−763
9


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*


 5(E)
−265
−1340
−52
1376
−1572
−1189
113
−1125
1345
−1287
−496
99
−1321
505
198
−218
−205
597
−1598
−1032
10


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*


 6(S)
256
−397
−1014
−830
−1841
−646
−862
−1443
−767
−1740
−963
−568
−1249
−651
−1007
2267
1586
−862
−2080
−1672
11


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*


 7(M)
−990
−889
−2630
157
−513
−2514
−1346
1309
−1767
820
3683
−1898
−2491
−1496
−1799
−1589
−925
150
−1336
−1041
12


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 8(E)
588
−1875
−194
1536
−2188
−1373
−59
−1931
957
−1890
−977
904
292
393
−162
483
−372
−1495
−2070
−1391
13


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 9(N)
−514
−1116
1207
−315
447
−1650
−304
−778
−224
825
−277
1457
−1738
−123
−618
−627
−454
−603
−1186
763
14


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 10(N)
−815
−1190
−1360
−922
−904
−1967
−797
−442
−670
381
1700
3009
−2099
−654
−934
−1051
−791
−445
−1490
−979
15


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 11(K)
−1530
−2498
−1722
−855
−3141
−2246
−428
−2627
2828
−2404
−1656
−927
662
−2
2047
−1421
−1337
−2324
−2357
−2081
16


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 12(Y)
−872
−1887
−861
−290
−1369
−1801
1662
−1797
325
−1793
−1031
893
−1876
56
2219
−812
−780
−1514
−1565
2287
17


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 13(S)
−830
−1586
−1471
−1099
−2717
−1642
−1010
−2479
−266
−2518
−1746
−1065
−2069
−676
1822
2748
−1000
−1950
−2597
−2189
18


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 14(Q)
−851
−2131
−775
−153
−2554
−1735
−211
−2205
1908
−2094
−1244
−386
−1802
2254
974
1001
−747
−1819
−2181
−1667
19


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 15(T)
−405
−1258
−618
−100
−1490
−1466
1158
−1121
1
−1299
−514
578
−1607
65
−433
960
1849
343
−1677
−1143
20


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 16(I)
−1772
−1325
−4307
−3877
−1405
−3993
−3383
2935
−3705
820
−217
−3632
−3761
−3400
−3682
−3260
−1742
2033
−2838
−2525
21


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 17(T)
−1018
−1329
−2004
−1771
−409
−1993
−1000
−1256
−1512
−1464
−966
−1543
−2367
−1428
−1638
−1257
3050
−1090
−1012
2448
22


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 18(Q)
−1509
−3056
1970
44
−3310
−1666
−896
−3242
−877
−3158
−2439
−322
−2123
3562
−1493
−1259
−1550
−2779
−3260
−2446
23


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 19(D)
−1006
−2199
2178
−88
−3159
1997
−936
−2974
−948
−2977
−2174
−382
−1960
−589
−1571
1295
−1157
−2369
−3178
−2430
24


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 20(M)
445
−796
−1082
−521
−841
−1643
−412
−403
−370
−692
2213
−646
536
1166
−698
−630
660
831
−1204
−767
25


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 21(Q)
741
−990
−1025
−507
−1249
−1551
−519
−720
−357
−1062
−345
−635
−1739
1770
−713
−589
1576
1129
−1559
−1097
26


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 22(R)
−1753
−2648
−2072
−1047
−3365
−2405
−452
−2782
1989
−2495
−1773
−1062
−2379
2402
2643
−1629
−1506
−2504
−2397
−2190
27


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 23(S)
−330
−1010
−1820
−1628
−2778
−1229
−1652
−2481
−1592
−2691
−1841
−1273
2130
−1426
−1834
2449
1034
−1716
−2961
−2594
28


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 24(P)
1882
−1119
−2231
−2302
−3062
−1360
−2209
−2710
−2339
−3013
−2243
−1676
3304
−2117
−2409
−742
−918
−1916
−3263
−3022
29


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 25(N)
969
−1230
−1066
−915
−2593
−1313
−1196
−2242
−1033
−2447
−1626
3197
−1850
−898
−1392
−582
1155
−1644
−2736
−2256
30


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 26(R)
−1847
−2640
−2014
−1161
−3282
−2428
−579
−2818
687
−2553
−1869
−1165
−2462
2447
3181
−1746
−1630
−2555
−2447
−2228
31


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 27(A)
3048
−932
−2480
−2533
−3075
−1200
−2274
−2765
−2501
−3071
−2221
−1658
−1948
−2205
−2512
1225
−739
−1842
−3322
−3078
32


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 28(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
33


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 29(Y)
−1674
−1506
−2863
−2464
596
−2872
2251
−972
−2024
2197
−552
−1986
−2876
−1739
−1988
−1987
−1601
−1002
−95
2332
34


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 30(Y)
−2013
−2305
−2428
−1781
−328
−2709
−654
−2240
−258
−2064
−1626
−1631
−2788
−899
2789
−2017
−1896
−2130
−857
3434
35


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 31(A)
2822
−1031
−2418
−2539
−3226
1898
−2364
−2941
−2626
−3229
−2379
−1722
−2026
−2302
−2634
−654
−848
−1983
−3415
−3226
36


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 32(I)
−1247
−941
−3569
−3039
−1082
−3101
−2185
2227
−2763
766
−76
−2700
−3050
−2469
−2697
−2253
1322
1974
−1988
−1633
37


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 33(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
38


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 34(F)
−1511
−1236
−3511
−3017
2747
−2982
−1069
−260
−2651
992
2737
−2407
−2904
−2088
−2418
−2099
−1434
−489
−537
2056
39


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 35(Q)
−576
−1869
−401
92
−2232
831
−173
−1930
1505
−1913
−1042
−186
−1620
1653
−51
−482
1346
−1534
−2098
−1490
40


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 36(D)
−1352
−3066
3028
1349
−3303
−1566
−724
−3141
1155
−3043
−2267
−165
−1991
−354
−1350
−1086
−1368
−2659
−3221
−2356
41


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 37(E)
−1507
−3288
2042
2762
−3520
515
−853
−3401
−981
−3296
−2566
−182
−2064
−503
−1753
−1209
−1553
−2895
−3486
−2547
42


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 38(D)
−1445
−2778
3529
−53
−3524
−1590
−1129
−3476
−1367
−3459
−2774
−396
−2156
−825
−2122
554
−1609
−2880
−3582
−2717
43


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 39(F)
−2658
−2176
−4213
−4000
3815
−3933
−1352
−531
−3638
1121
−19
−3184
−3709
−2820
−3296
−3219
−2579
−1037
−601
403
44


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 40(D)
−684
−2193
1738
1460
−2494
−1437
−249
−2257
1694
−2199
−1308
−62
−1637
185
−450
−531
633
−1808
−2374
−1657
45


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 41(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
46


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 42(P)
1882
−1119
−2231
−2302
−3062
−1360
−2209
−2710
−2339
−3013
−2243
−1676
3304
−2117
−2409
−742
−918
−1916
−3263
−3022
47


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 43(I)
−1006
−992
−2347
−1784
−650
−2452
−1256
2372
−1386
77
2213
−1720
−2455
2030
−1490
−1528
−946
106
−1441
−1111
48


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 44(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
49


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 45(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
50


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 46(I)
−1759
−1303
−4330
−3968
−1751
−4051
−3743
3027
−3837
−597
−528
−3729
−3875
−3688
−3910
−3369
−1751
2438
−3259
−2819
51


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 47(V)
1736
−1012
−3546
−3078
−1377
−3073
−2434
2052
−2843
−608
−331
−2754
−3122
−2619
−2855
−2270
−1277
2193
−2333
−1941
52


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 48(N)
−686
−1511
−702
−806
−2927
−1386
−1339
−2841
−1264
−2950
−2137
2702
−1979
−1062
−1648
2444
−971
−2105
−3054
−2475
53


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 49(M)
−411
−857
−1800
−1434
−1528
1914
−1202
−1029
−1247
−1347
2989
−1217
−1912
−1119
−1444
−676
1550
−767
−1922
−1539
54


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 50(W)
−782
−1258
793
−683
1193
346
2051
−932
−556
−1092
−441
−798
−1993
−426
−909
−904
−720
−779
3163
1546
55


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 51(W)
1009
−798
−1470
−935
−463
−1773
−545
−460
−751
−736
−66
−943
−1904
−606
−1002
1604
−507
−322
2535
1521
56


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 52(D)
−1137
−2711
2125
1647
−2995
−1523
−617
−2786
−528
−2743
−1933
−150
−1897
−234
−1165
−924
2117
−2331
−2948
−2141
57


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 53(I)
−599
−1102
−1031
−829
−1522
1429
−927
2119
−880
−1369
−699
1692
−1938
−759
−1188
−799
−698
−689
−1887
−1419
58


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 54(T)
−666
−1412
−954
−984
−2702
−1428
−1357
−2418
−1208
−2650
−1886
2293
−2000
−1101
−1519
−787
2967
−1835
−2866
−2360
59


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 55(P)
−632
−1230
−2074
−2144
−2996
−1453
−2116
−2631
−2128
−2928
−2213
−1658
3610
−2006
−2221
−852
1302
−1931
−3185
−2917
60


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 56(C)
−2476
5735
−4102
−4358
−3712
−2763
−3545
−3518
−4167
−3859
−3569
−3631
−3363
−4030
−3832
−2793
−2860
−3158
−3464
−3718
61


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 57(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
62


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 58(M)
672
−918
−3119
−2578
−742
−2668
−1734
1807
−2263
16
3713
−2271
−2704
−1960
−2216
−1806
−1058
493
−1612
−1306
63


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 59(H)
−1525
−2164
−1235
−1346
−2509
2296
4235
−3172
−1516
−3178
−2523
−1448
−2541
−1520
−1760
−1591
−1741
−2656
−2681
−2065
64


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 60(L)
−2478
−2009
−4717
−4196
−568
−4424
−3262
1334
−3887
2824
604
−4085
−3872
−3088
−3590
−3717
−2380
−199
−2217
−2207
65


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 61(H)
−682
−2191
1015
275
−2485
396
2379
−2251
62
−2197
−1307
1826
−1636
1527
−480
−529
−641
−1803
−2375
−1654
66


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 62(D)
−575
−1920
1979
184
−2299
94
−242
−2029
114
−2023
−1144
−120
−1608
186
1063
−469
1413
−1605
−2229
−1561
67


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 63(L)
−2618
−2139
−4597
−4163
2144
−4285
−2334
−83
−3854
2690
538
−3771
−3806
−2950
−3488
−3563
−2505
−751
−1442
−808
68


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 64(A)
2657
−1033
−2408
−2532
−3233
2193
−2364
−2950
−2626
−3237
−2386
−1719
−2027
−2301
−2635
−655
−850
−1988
−3420
−3231
69


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 65(K)
−443
−1857
958
270
−2158
−1393
−66
−1890
1839
−442
−957
−36
−1499
1204
−132
616
−382
−1469
−2048
−1383
70


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 66(C)
605
1553
739
−17
−1374
−1488
−182
260
969
−203
−397
−263
−1573
159
691
−426
−331
−761
−1567
−1032
71


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 67(A)
2327
−956
−3193
−2728
−1289
−2677
−2114
1664
−2485
−601
−288
−2403
−2839
−2263
−2523
−1871
−1126
1617
−2143
−1765
72


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 68(K)
−532
−1656
−490
1321
−1891
−1527
−172
−124
2206
−1591
−782
−223
−1619
237
−106
−482
−464
−98
−1904
−1326
73


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 69(H)
384
−1854
936
889
−2165
−1363
1498
−1909
1111
−1866
−948
1091
−1464
421
−131
−284
−342
−69
−2043
−1364
74


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 70(G)
1823
−932
−2330
−2313
−3120
2511
−2158
−2865
−2331
−3098
−2209
−1563
−1912
−2032
−2419
1138
−706
−1883
−3328
−3077
75


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 71(V)
−1760
−1333
−4244
−3789
−1262
−3902
−3190
1495
−3588
1270
−96
−3536
−3677
−3238
−3534
−3148
−1725
2865
−2654
−2373
76


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 72(W)
−1054
−2172
−1112
−403
−2566
−1917
−286
−2196
2516
−2095
−1292
1183
−1958
140
1333
−959
−922
−1867
2591
−1720
77


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 73(D)
611
−1995
1525
937
−2295
−1400
−148
−2043
211
−2006
−1106
−37
−1553
1420
−312
−408
1235
−1609
−2193
−1499
78


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 74(A)
2716
−902
−2380
−2205
−2799
−1197
−1975
−2459
−2081
−2736
−1895
−1520
−1895
−1844
−2201
1191
1299
−1669
−3045
−2758
79


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 75(G)
−1709
−2833
2424
−409
−3781
2819
−1457
−3777
−1728
−3733
−3076
−739
−2389
−1180
−2441
−1557
−1893
−3158
−3660
−3038
80


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−212
−2909
−8150
−273
−2534
−701
−1378
*
*


 76(A)
2529
−1119
−2614
−2330
−1245
−1983
−1829
−377
−2042
1435
−341
−1937
−2411
−1873
−2088
−1266
−1059
−397
−2063
−1713
82


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 77(W)
−472
−361
−2421
−1812
−298
−1979
−826
1164
−1486
−143
2485
873
−2028
−1185
−1426
−1048
−412
1116
2999
−454
83


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 78(P)
−1198
−1737
−2187
−2394
−3665
2006
−2550
−3630
−2743
−3756
−3008
−2052
3474
−2495
−2835
−1401
−1593
−2736
−3511
−3519
84


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 79(Q)
−999
−1075
−2106
−1568
−726
−2370
−1175
83
−1185
1373
218
−1566
−2400
2445
−1340
−1445
−946
1441
−1501
−1146
85


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 80(Q)
−885
−779
−2609
−2018
−481
−2414
−1253
1645
−1736
799
1924
−1827
−2405
2262
−1752
−1484
−821
802
−1240
−935
86


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 81(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
87


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 82(G)
−998
−2100
−120
−175
−2567
2528
2174
−2558
−587
−2583
−1806
1422
−1966
−461
−1038
−925
−1088
−2095
−2657
−1948
88


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 83(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
89


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 84(I)
−1286
−1279
−2907
−2683
−1446
−2549
−2198
3290
−2407
−726
−534
−2386
1172
−2299
−2437
−1895
−1392
283
−2302
−1913
90


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 85(T)
−493
−1105
−2189
−2267
−3101
1880
−2196
−2791
−2334
−3081
−2269
−1649
−2058
−2099
−2410
−719
3135
−1948
−3282
−3046
91


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 86(V)
−1750
−1296
−4319
−3957
−1765
−4038
−3733
2364
−3826
−619
−543
−3716
−3869
−3685
−3902
−3354
−1743
3012
−3265
−2817
92


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 87(S)
923
−962
−2348
−2422
−3132
−1207
−2248
−2850
−2440
−3140
−2285
−1624
−1954
−2158
−2477
3171
−758
−1896
−3362
−3103
93


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 88(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
94


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 89(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
95


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 90(I)
−1880
−1493
−4193
−3724
−953
−3837
−2980
3251
−3420
257
2372
−3485
−3608
−3005
−3310
−3087
−1840
617
−2373
−2155
96


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 91(S)
2150
−939
−2407
−2415
−3075
−1197
−2205
−2781
−2384
−3065
−2205
−1613
−1936
−2105
−2436
2652
−729
−1850
−3306
−3049
97


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 92(M)
−979
−1455
−1242
−1122
−1434
−1860
−1131
−1171
−974
−1285
4091
2176
−2226
−1017
−1187
−1166
−1086
−1063
−1929
−1345
98


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 93(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
99


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 94(T)
−959
−1691
−1249
−949
−2563
−1747
−929
−2093
1282
−2263
−1554
−995
−2115
−600
−354
−1037
3152
−1726
−2494
−2098
100


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 95(E)
−572
−1860
−208
2213
−2107
−1461
−191
−1808
199
−116
−983
−127
318
1199
−269
−475
−517
−1448
−2078
−1441
101


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 96(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
102


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 97(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
103


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 98(R)
−2097
−2786
−2688
−1415
−3622
−2625
−555
−2964
2585
−2627
−1957
−1318
−2577
−137
3015
−1979
−1791
−2732
−2469
−2363
104


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


 99(Y)
−3615
−2706
−4169
−4413
2626
−4044
−396
−2535
−3993
−1939
−1985
−2747
−3930
−2852
−3446
−3296
−3494
−2686
347
4252
105


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


100(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
106


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


101(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
107


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


102(V)
−1381
−1065
−3714
−3252
−1453
−3300
−2646
1872
−3023
−615
−373
−2949
−3287
−2816
−3039
−2506
1346
2750
−2489
−2087
108


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


103(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
109


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


104(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
110


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


105(E)
−1719
−3572
2596
2779
−3767
−1632
−993
−3700
−1241
−3578
−2920
−234
−2167
−666
−2090
−1380
−1789
−3182
−3742
−2756
111


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


106(V)
−1746
−1296
−4308
−3946
−1757
−4020
−3712
2190
−3811
−614
−539
−3702
−3858
−3667
−3884
−3336
−1740
3098
−3250
−2803
112


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


107(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
113


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


108(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
114


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


109(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
115


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


110(S)
−352
2942
−2955
−2957
−2876
−1254
−2382
−2573
−2692
−2927
−2128
−1827
−2001
−2405
−2607
3103
−778
−1757
−3171
−2911
116


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


111(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
117


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


112(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
118


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


113(T)
1556
−936
−2493
−2457
−2805
−1256
−2159
−2210
−2319
−2681
−1932
−1656
−1974
−2089
−2352
−598
3235
−1547
−3111
−2847
119


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


114(C)
1784
2119
−2013
−1532
−1093
−1580
−1089
−436
−1322
−937
−273
1093
−1932
−1127
−1472
−748
−515
1585
−1536
−1163
120


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


115(M)
1831
2019
−2596
−2038
−605
−1979
−1126
244
−1727
−359
2501
−1655
−2145
−1435
−1683
−1106
−557
1087
−1153
−804
121


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


116(Q)
−987
−2211
−43
−62
−2833
2229
−691
−2616
−407
−2604
−1797
1197
−1917
2260
−858
−880
−1045
−2139
−2772
−2099
122


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


117(G)
2313
−1042
−2391
−2526
−3250
2601
−2372
−2972
−2637
−3257
−2407
−1721
−2032
−2310
−2646
−662
−859
−2003
−3434
−3247
123


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


118(Q)
−914
−2350
−48
1661
−2621
−1571
2504
−2400
68
−2331
−1486
−201
−1796
2646
−351
−754
−865
−1984
−2463
−1787
124


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


119(W)
−517
−1294
−733
−183
−1062
−1605
−234
−1037
19
−1207
−456
1435
−1690
33
756
411
−454
−819
3340
1286
125


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


120(M)
410
−469
−2417
−1828
−341
−2041
−897
195
−1513
−156
3130
−1534
−2102
−1230
−1484
−1117
−507
954
−894
2253
126


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


121(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
127


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


122(G)
2142
−930
−2334
−2298
−3100
2237
−2139
−2842
−2302
−3074
−2187
−1557
−1909
−2010
−2397
1136
−701
−1871
−3308
−3053
128


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


123(V)
−1514
−1144
−3950
−3459
1821
−3487
−2577
2274
−3208
−209
−87
−3112
−3362
−2864
−3118
−2680
−1476
2426
−2194
−1786
129


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


124(V)
−1743
−1294
−4292
−3873
−1511
−3988
−3433
2287
−3712
598
−319
−3626
−3774
−3456
−3716
−3260
−1717
2790
−2931
−2577
130


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


125(A)
2911
−954
−2808
−2665
−2115
−1577
−2196
−575
−2445
−1646
−1202
−1906
−2208
−2218
−2451
−901
−876
1294
−2727
−2394
131


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


126(I)
−1764
−1323
−4298
−3936
−1668
−3994
−3655
3337
−3783
−508
−462
−3689
−3838
−3608
−3835
−3311
−1759
1847
−3164
−2747
132


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


127(G)
−1157
−1705
−2169
−2375
−3654
3021
−2534
−3611
−2730
−3741
−2984
−2024
2418
−2475
−2826
−1361
−1555
−2705
−3513
−3509
133


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


128(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
134


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


129(C)
−2476
5735
−4102
−4358
−3712
−2763
−3545
−3518
−4167
−3859
−3569
−3631
−3363
−4030
−3832
−2793
−2860
−3158
−3464
−3718
135


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


130(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
136


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


131(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
137


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


132(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
138


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


133(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
139


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


134(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
140


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


135(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
141


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


136(A)
2180
−935
−2286
−2196
−3057
1098
−2058
−2796
−2174
−3021
−2134
−1516
−1898
−1906
−2302
2146
−689
−1849
−3256
−2983
142


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


137(M)
−1799
−1433
−4142
−3579
−669
−3668
−2608
1558
−3293
1235
3799
−3296
−3401
−2717
−3088
−2843
−1726
1156
−2002
−1868
143


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


138(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
144


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


139(A)
3103
−1036
−2445
−2572
−3222
1051
−2380
−2930
−2650
−3226
−2381
−1739
−2034
−2327
−2648
−664
−857
−1981
−3412
−3228
145


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


140(M)
−2325
−1891
−4598
−4012
−498
−4222
−3013
1242
−3722
1864
3929
−3855
−3711
−2910
−3414
−3439
−2215
−299
−2076
−2098
146


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


141(A)
3103
−1036
−2445
−2572
−3222
1051
−2380
−2930
−2650
−3226
−2381
−1739
−2034
−2327
−2648
−664
−857
−1981
−3412
−3228
147


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


142(R)
−1588
−2442
−1399
−953
−3069
−2171
−708
−2795
373
−2625
−1916
1858
−2357
−324
3294
−1520
−1505
−2453
−2523
−2186
148


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


143(M)
−1448
−1256
−3396
−2819
−474
−3024
−1923
175
−2473
2225
2756
−2574
−2922
−2063
−2375
−2153
952
−151
−1599
−1410
149


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


144(N)
−1662
−3306
2055
78
−3621
−1643
−1040
−3622
−1272
−3531
−2870
3477
−2182
−724
−2071
−1371
−1757
−3092
−3633
−2700
150


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


145(I)
−1066
−921
−2828
−2239
−1041
−2675
−1601
2235
−1668
−455
−92
−2067
−2692
−1688
1701
−1795
−1024
1960
−1771
−1396
151


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


146(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
152


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


147(S)
1568
−940
−2267
−2192
−3082
1101
−2068
−2826
−2185
−3049
−2159
−1515
−1901
−1915
−2313
2603
−694
−1866
−3279
−3006
153


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


148(I)
−1880
−1492
−4195
−3728
−963
−3841
−2991
3272
−3425
246
2277
−3490
−3613
−3014
−3317
−3092
−1841
628
−2385
−2163
154


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


149(F)
−2204
−1797
−3724
−3473
3206
−3383
−628
−1077
−3092
−746
3167
−2502
−3309
−2372
−2792
−2535
−2120
−1245
28
2460
155


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


150(V)
1265
−1028
−3200
−2994
−1833
−2150
−2480
417
−2771
−1122
−818
−2349
−2640
−2559
−2766
−1464
−1118
3028
−2700
−2325
156


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


151(Y)
−3482
−2868
−3701
−3919
238
−3552
−1112
−3000
−3638
−2516
−2526
−3027
−3772
−3101
−3341
−3418
−3527
−3071
−441
4711
157


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


152(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
158


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


153(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
159


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


154(T)
−359
−976
−2225
−2229
−2900
−1242
−2074
−2560
−2170
−2875
−2064
−1561
−1958
−1969
−2247
1110
3375
−1760
−3152
−2850
160


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


155(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
161


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


156(H)
861
−1924
−384
1010
−2260
−1477
1787
−1974
1769
−1918
−1022
−120
−1566
362
697
−417
−459
−1557
−2073
−1446
162


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


157(P)
−655
−1502
−711
−557
−2204
−1463
2143
−2122
−586
−2233
−1445
−688
2941
−560
−941
855
−805
−1657
−2369
−1763
163


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


158(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
164


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


159(H)
−744
−2193
−114
1118
−2513
−1512
2486
−2252
1178
−2183
−1308
2230
−1689
180
−233
−598
−687
−1823
−2335
−1670
165


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


160(W)
−2672
−2139
−3850
−3748
941
−3611
−469
−1691
−3306
1047
−1217
−2551
−3534
−2514
−2960
−2788
−2577
−1799
4205
3466
166


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


161(K)
386
−1981
779
279
−2295
−1403
−114
−2043
2059
−1991
−1082
941
−1536
1263
−211
−384
−457
−1602
−2161
−1476
167


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


162(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
168


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


163(K)
−1144
−2365
−912
2048
−2856
−1912
−326
−2459
2267
−2295
−1482
−556
−1989
108
1334
−1013
−1014
−2093
−2324
−1881
169


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


164(D)
−1091
−2610
2941
174
−2957
−1527
−595
−2750
1084
−2696
−1877
−176
−1885
−206
−1006
740
−1098
−2288
−2880
−2105
170


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


165(L)
−2387
−1922
−4674
−4155
−617
−4366
−3250
1889
−3865
2650
558
−4023
−3847
−3098
−3586
−3647
−2296
−38
−2247
−2224
171


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


166(N)
−1021
−2427
1806
133
−2870
−1499
−635
−2647
−521
−2640
−1825
2171
−1874
−255
−1124
−860
2122
−2184
−2853
−2090
172


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


167(I)
−1830
−1390
−4327
−3873
−1210
−3994
−3274
2967
−3678
1259
−30
−3633
−3730
−3283
−3604
−3249
−1791
1570
−2661
−2417
173


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


168(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
174


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


169(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
175


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


170(A)
2440
−824
−2371
−2082
−1993
−1344
−1704
−1264
−1899
−1832
−1137
−1517
−1946
−1674
−2005
1075
−641
1474
−2390
−2055
176


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


171(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
177


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


172(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
178


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


173(A)
2966
−1031
−2429
−2551
−3222
1544
−2368
−2934
−2633
−3225
−2377
−1727
−2028
−2309
−2637
−656
−850
−1980
−3412
−3224
179


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


174(V)
−1769
−1342
−4255
−3793
−1216
−3901
−3162
1633
−3589
1486
−51
−3537
−3667
−3214
−3518
−3143
−1731
2692
−2609
−2345
180


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


175(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
181


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


176(Q)
−729
−2116
−413
1096
−2484
−1587
1599
−2186
1695
−2094
−1219
−223
−1698
2418
90
−599
−649
−1770
−2213
−1615
182


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


177(W)
−1652
−1707
−2340
−1879
1996
−2733
2013
−1398
1758
−1386
−938
−1641
−2751
−1364
−1762
−1780
−1577
−1325
3577
2136
183


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


178(T)
−421
−753
−1251
−704
−846
−1670
−535
894
−548
−690
−1
1376
−1791
−421
−846
373
1461
858
−1236
−812
184


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


179(H)
1498
−1593
−504
15
−1895
−1484
2279
−1559
1119
−1640
−810
−242
−1611
194
−171
−462
815
−1231
−1914
−1340
185


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


180(G)
−1515
−2130
−1298
−1450
−2658
3285
2212
−3276
−1691
−3291
−2638
−1524
−2562
−1662
−1925
−1600
−1764
−2713
−2804
−2234
186


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


181(K)
−528
−2010
1346
1082
−2329
−1408
−118
−2080
1475
−2018
−1108
1161
−1543
331
1052
−394
−471
−1632
−2181
−1494
187


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


182(M)
−1894
−1521
−4170
−3679
−840
−3793
−2866
2827
−3360
375
3445
−3437
−3555
−2902
−3223
−3028
−1846
470
−2249
−2059
188


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


183(T)
−670
−1758
1731
−141
−2591
−1399
−691
−2319
−499
−2384
−1543
−387
−1786
−316
−1016
1576
2044
−1811
−2624
−1981
189


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


184(E)
345
−2074
925
1994
−2378
−1408
−177
−2135
922
−2084
−1183
−38
641
264
−356
−444
−536
−1690
−2261
−1556
190


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


185(E)
−1493
−2900
93
3174
−2903
−1743
1987
−3042
−646
−2957
−2238
−411
−2146
−506
−1121
−1272
−1503
−2629
−2905
−2134
191


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


186(D)
−1293
−2959
2673
2121
−3219
−1546
−713
−3043
−707
−2974
−2191
−158
−1967
−342
−1394
−1043
701
−2567
−3172
−2311
192


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


187(F)
−1137
−905
−3250
−2707
2365
−2647
−1016
−34
−2336
1239
267
−2150
−2626
−1861
−2133
−1752
−1069
1461
−599
1844
193


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


188(K)
−479
−1713
−409
1031
−1925
−1467
1755
−1650
1844
−349
−827
−140
−1556
319
−75
−403
−411
−1301
−1900
843
194


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


189(G)
433
−2144
52
1047
−2717
2303
−615
−2467
−442
−2482
−1655
1123
−1828
−233
−995
−763
−923
−2000
−2710
−2005
195


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


190(V)
−1752
−1320
−4254
−3806
−1311
−3916
−3232
1701
−3614
1188
−140
−3551
−3693
−3280
−3568
−3166
−1718
2833
−2703
−2409
196


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


191(E)
−1199
−1750
−734
2668
−1820
−2038
−1068
1892
−867
−1273
−897
−922
−2295
−797
−1238
−1340
−1197
−426
−2325
−1789
197


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


192(C)
−1182
3528
−1398
−620
−2541
−2038
−358
−2093
1181
−2037
−1272
−747
−2070
1553
2213
−1123
−1038
−1817
−2142
−1774
198


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


193(N)
−1478
−2527
−261
−403
−2011
−1837
2032
−2925
−735
−2845
−2195
3635
−2259
−721
−1085
−1352
−1546
−2522
−2307
−1431
199


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


194(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
200


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


195(C)
−1220
4911
−3609
−3314
−1440
−2525
−2482
1565
−2922
−706
−544
−2678
−2896
−2710
−2836
−1869
−1375
379
−2371
−1957
201


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


196(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
202


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


197(G)
−477
−1115
−1983
−2189
−3315
3154
−2272
−3172
−2506
−3387
−2522
−1599
−2042
−2177
−2583
1217
−905
−2130
−3477
−3225
203


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


198(A)
1653
−1347
−705
−249
−1969
−1385
−477
−1629
−159
−1759
−935
−434
1285
1404
−586
−450
1019
−1243
−2070
−1522
204


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


199(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
205


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


200(S)
1870
−938
−2270
−2183
−3068
1488
−2056
−2810
−2168
−3032
−2144
−1511
−1898
−1901
−2300
2236
−690
−1857
−3265
−2990
206


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


201(C)
−2476
5735
−4102
−4358
−3712
−2763
−3545
−3518
−4167
−3859
−3569
−3631
−3363
−4030
−3832
−2793
−2860
−3158
−3464
−3718
207


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


202(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
208


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


203(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
209


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


204(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
210


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


205(Y)
−3590
−2700
−4146
−4379
2092
−4028
−404
−2517
−3963
−1928
−1973
−2744
−3921
−2845
−3431
−3284
−3474
−2669
336
4423
211


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


206(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
212


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


207(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
213


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


208(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
214


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


209(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
215


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


210(M)
−2355
−1988
−4343
−3834
−504
−4051
−2868
105
−3385
1451
4460
−3680
−3671
−2806
−3171
−3327
−2274
−474
−2039
−1925
216


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


211(S)
2150
−939
−2407
−2415
−3075
−1197
−2205
−2781
−2384
−3065
−2205
−1613
−1936
−2105
−2436
2652
−729
−1850
−3306
−3049
217


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


212(S)
−344
−979
−2190
−2162
−2959
−1227
−2042
−2651
−2116
−2934
−2100
−1526
−1941
−1909
−2222
2940
1775
−1804
−3187
−2882
218


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


213(A)
3048
−932
−2480
−2533
−3075
−1200
−2274
−2765
−2501
−3071
−2221
−1658
−1948
−2205
−2512
1225
−739
−1842
−3322
−3078
219


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


214(I)
−1924
−1546
−4067
−3658
2312
−3663
−2081
3030
−3367
150
99
−3197
−3492
−2821
−3179
−2894
−1877
293
−1445
−692
220


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


215(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
221


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


216(A)
2389
−814
−2506
−2162
−1696
−1545
−1698
−499
−1942
−1398
−813
−1640
−2076
−1723
−2027
−806
1148
1559
−2200
−1856
222


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


217(M)
−2576
−2118
−4725
−4165
−461
−4430
−3165
99
−3811
2513
3454
−4075
−3839
−2978
−3488
−3704
−2457
−591
−2111
−2145
223


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


218(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
224


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


219(M)
−2313
−1968
−4258
−3765
−518
−3966
−2806
98
−3289
1292
4523
−3599
−3636
−2769
−3097
−3249
−2243
−457
−2026
−1874
225


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


220(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
226


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


221(L)
−2631
−2159
−4786
−4228
−462
−4506
−3231
96
−3878
2828
2482
−4157
−3880
−3016
−3541
−3793
−2509
−608
−2134
−2182
227


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


222(P)
−1501
−1778
−2473
−2371
−1710
−2311
−2045
−1321
−2060
827
−1068
−2173
3594
−2082
−2130
−1799
−1699
−1373
−2373
−1942
228


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


223(Y)
−1068
−1670
−865
−836
−631
1198
−767
−1828
−1059
−1914
−1304
692
−2203
−906
−1387
−1136
−1163
−1566
−1185
3670
229


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


224(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
230


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


225(S)
1172
−954
−2367
−2422
−3120
−1204
−2237
−2835
−2426
−3122
−2265
−1621
−1948
−2145
−2467
3107
−749
−1884
−3349
−3092
231


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


226(S)
−342
−975
−2176
−2124
−2912
−1229
−2003
−2594
−2067
−2878
−2048
−1510
−1936
−1866
−2184
2553
2492
−1773
−3143
−2833
232


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


227(M)
−720
−1440
−710
−343
−1228
−1693
2436
−1209
−132
−1364
3099
1904
−1852
−183
−458
−776
−680
−1004
−1540
−890
233


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


228(P)
2240
−1100
−2241
−2293
−3037
−1346
−2188
−2683
−2317
−2986
−2210
−1663
3041
−2093
−2391
−722
−895
−1893
−3243
−2998
234


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


229(A)
2958
−1235
−1299
−1377
−2868
−1345
−1673
−2580
−1661
−2843
−2054
1555
−1995
−1468
−1921
−715
−888
−1871
−3064
−2630
235


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


230(E)
−509
−1046
−884
1564
−1116
−1669
−441
−485
−283
250
−206
−577
689
−200
−656
−670
−459
1290
−1467
−995
236


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


231(D)
−1203
−2412
2595
−117
−3286
−1536
−1057
−3176
−1165
−3186
−2436
−428
−2068
−736
−1824
2377
−1366
−2578
−3334
−2552
237


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


232(Q)
954
−1983
−100
971
−2337
177
−267
−2067
81
−2060
−1189
−125
−1637
2600
−418
−514
−597
−1649
−2268
−1597
238


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


233(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
239


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


234(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
240


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


235(R)
377
−1802
−415
988
−2095
−1474
−95
−1786
1452
−1785
−911
−135
−1560
343
1555
−409
−431
376
−1986
−1375
241


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


236(D)
1083
−1565
2662
−244
−1941
−1573
−679
612
−527
−1651
−980
−490
−1869
−358
−1003
−771
−766
−903
−2208
−1633
242


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


237(E)
−1225
−2868
1894
1948
−3149
−1532
−671
−2975
−630
−2902
−2101
−150
−1935
−293
−1299
1884
−1241
−2496
−3093
−2248
243


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


238(C)
1375
3262
−2620
−2108
−827
−1866
−1267
1631
−1811
−599
−10
−1674
−2137
−1531
−1786
−1034
790
249
−1361
−1010
244


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


239(E)
635
−1796
1055
1761
−2018
−1464
−263
1191
28
−1767
−946
−148
−1637
135
−481
−520
−553
−1300
−2077
−1441
245


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


240(E)
593
−2044
−252
2548
−2437
−1542
−329
−2133
151
−2120
−1274
−244
−1738
89
946
−646
−717
−1734
−2305
−1686
246


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


241(S)
1884
−835
−1962
−1576
−1634
−1436
−1320
1041
−1409
−1453
−781
−1293
−1922
−1241
−1606
1973
−597
−669
−2036
−1656
247


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


242(G)
2267
−1043
−2388
−2526
−3253
2642
−2373
−2975
−2639
−3260
−2410
−1722
−2033
−2311
−2648
−663
−860
−2005
−3436
−3250
248


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


243(R)
−876
−2087
−829
1490
−2474
−1766
−229
−2106
1269
−44
−1198
−424
−1829
205
2225
−775
−768
−1753
−2143
−1647
249


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


244(V)
2339
−967
−2970
−2766
−1878
−1847
−2252
32
−2541
−1299
−918
−2087
−2399
−2316
−2545
−1157
−971
2345
−2605
−2251
250


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


245(I)
−1827
−1398
−4307
−3831
−1099
−3939
−3142
2286
−3619
1835
69
−3579
−3671
−3177
−3511
−3178
−1781
1918
−2524
−2310
251


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


246(V)
−1178
−1448
−1943
−1452
−1776
−2261
−1140
−227
1866
−1260
−816
−1444
−2448
−902
−540
−1496
−1176
2697
−2161
−1764
252


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


247(E)
−508
−1976
840
1547
−2280
−1393
−117
−2029
1400
−1984
−1077
1158
−1531
330
−253
−378
−454
262
−2163
−1471
253


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


248(M)
1703
−991
−2901
−2342
−528
−2567
−1550
166
−2031
1544
2668
−2104
−2591
−1715
−2010
−1685
−1052
−12
−1442
−1177
254


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


249(I)
−1947
−1516
−4385
−3885
−916
−4013
−3118
2193
−3656
2186
257
−3656
−3687
−3109
−3494
−3250
−1889
1383
−2397
−2258
255


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


250(E)
−1322
−2647
−272
2491
−3071
−1811
−576
−2759
2306
−2633
−1854
−464
−2066
−175
−177
−1144
−1256
−2368
−2692
−2140
256


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


251(K)
−1395
−2059
−1711
−1014
−2215
−2218
−641
−1709
3021
−1652
2578
−1075
−2303
−282
287
−1423
−1283
−1603
−2159
−1803
257


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


252(D)
−1285
−2888
2677
176
−3210
1189
−737
−3047
−715
−2977
−2195
−190
−1979
2106
−1379
−1050
−1315
−2564
−3161
−2320
258


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


253(I)
−2073
−1632
−4434
−3975
−911
−4130
−3238
3164
−3706
1451
244
−3779
−3785
−3187
−3557
−3413
−2021
546
−2449
−2273
259


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


254(K)
−1570
−2144
−1887
−1191
−2098
−2363
−750
−1603
3034
938
−1112
−1231
−2436
−408
215
−1616
−1443
−1580
−2166
−1804
260


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


255(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
261


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


256(R)
−928
−1705
−1507
−1055
−2761
−1730
−896
−2490
−44
−2489
−1723
−1042
−2102
−543
2614
2258
−1053
−1998
−2546
−2158
262


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


257(D)
−1280
−2865
3154
175
−3194
−1547
−743
−3034
−728
−2971
−2194
−190
−1979
1342
−1391
553
−1316
−2552
−3161
−2317
263


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


258(I)
−1997
−1562
−4355
−3927
−1042
−4066
−3261
3343
−3654
937
97
−3718
−3783
−3239
−3555
−3364
−1959
702
−2549
−2295
264


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


259(M)
−2252
−1821
−4572
−3991
−530
−4164
−2990
2068
−3709
1993
3197
−3808
−3685
−2916
−3406
−3378
−2149
−172
−2084
−2091
265


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


260(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
266


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


261(R)
−2131
−2786
−2704
−1460
−3618
−2638
−587
−2976
1735
−2645
−1985
−1353
−2603
−173
3492
−2020
−1828
−2748
−2484
−2384
267


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


262(K)
−1349
−2635
−381
2083
−3083
−1857
−565
−2750
2690
−2612
−1837
−514
−2090
−161
−61
−1178
−1271
−2369
−2655
−2138
268


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


263(A)
2821
−932
−2451
−2472
−3065
−1198
−2233
−2763
−2434
−3056
−2201
−1633
−1940
−2147
−2468
1831
−730
−1840
−3305
−3055
269


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


264(F)
−2063
−1686
−4037
−3677
3437
−3644
−1706
2063
−3359
135
67
−3095
−3486
−2739
−3127
−2876
−2012
−83
−1038
−158
270


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


265(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
271


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


266(N)
−1662
−3306
2055
78
−3621
−1643
−1040
−3622
−1272
−3531
−2870
3477
−2182
−724
−2071
−1371
−1757
−3092
−3633
−2700
272


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


267(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
273


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


268(I)
−1760
−1307
−4325
−3962
−1735
−4042
−3726
3135
−3828
−579
−515
−3722
−3869
−3673
−3896
−3359
−1752
2276
−3240
−2806
274


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


269(T)
1428
−904
−2334
−2158
−2747
−1206
−1940
−2392
−2037
−2678
−1846
−1504
−1896
−1809
−2163
902
3001
−1635
−2999
−2705
275


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


270(V)
−1745
−1300
−4286
−3858
−1446
−3967
−3370
2358
−3688
852
−261
−3606
−3749
−3403
−3673
−3232
−1717
2643
−2856
−2524
276


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


271(V)
−1404
−1072
−3766
−3305
−1464
−3356
−2696
2276
−3080
−616
−379
−3001
−3325
−2870
−3091
−2563
1344
2521
−2516
−2113
277


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


272(M)
866
−1113
−2656
−2412
−1322
−1920
−1883
−487
−2061
−587
4451
−1950
−2387
−1928
−2078
−1220
−1053
−498
−2134
−1803
278


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


273(A)
2601
−957
−2898
−2711
−1943
−1740
−2211
−165
−2487
−1406
−1001
−2008
−2320
−2260
−2494
−1053
−929
1990
−2626
−2279
279


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


274(L)
−1171
−983
−3266
−2733
−796
−2795
−1888
590
−2418
2001
198
−2418
−2816
−2106
−2362
−1944
965
1777
−1724
−1426
280


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


275(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
281


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


276(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
282


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


277(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
283


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


278(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
284


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


279(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
285


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


280(A)
3134
−934
−2491
−2567
−3083
−1203
−2300
−2766
−2540
−3082
−2237
−1672
−1954
−2240
−2537
874
−747
−1844
−3333
−3093
286


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


281(V)
−984
−1045
−3169
−2909
−1709
−2304
−2404
531
−2643
−988
−697
−2378
−2722
−2480
−2661
−1601
1504
3014
−2588
−2201
287


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


282(L)
−2631
−2159
−4786
−4228
−462
−4506
−3231
96
−3878
2828
2482
−4157
−3880
−3016
−3541
−3793
−2509
−608
−2134
−2182
288


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


283(H)
−3205
−3079
−2723
−2890
−2110
−3046
5295
−4135
−2617
−3813
−3561
−2886
−3482
−2833
−2620
−3291
−3356
−3895
−2397
−1681
289


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


284(L)
−1623
−1338
−3726
−3164
−251
−3255
−1820
1373
−2808
2371
514
−2785
−3086
−2281
−2613
−2389
−1543
−161
−1311
1782
290


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


285(L)
−2333
−1873
−4640
−4127
−650
−4326
−3241
2176
−3843
2519
523
−3982
−3833
−3105
−3579
−3604
−2247
56
−2268
−2230
291


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


286(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
292


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


287(M)
−1886
−1507
−4178
−3693
−877
−3806
−2901
3008
−3380
335
3109
−3451
−3570
−2934
−3251
−3044
−1840
524
−2288
−2089
293


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


288(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
294


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


289(H)
−1490
−2484
−362
−476
−1816
−1880
4320
−2854
−684
−2770
−2133
2185
−2285
−728
−1000
−1377
−1550
−2475
−2146
−1255
295


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


290(A)
2439
−911
−2326
−2131
−2811
−1197
−1934
−2480
−2011
−2745
−1898
−1490
−1888
−1785
−2153
1898
1073
−1682
−3044
−2749
296


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


291(I)
2038
−985
−3388
−2919
−1320
−2893
−2277
2155
−2677
−587
−297
−2593
−2992
−2450
−2697
−2087
−1208
1681
−2229
−1846
297


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


292(G)
−1243
−2769
311
1902
−3172
1980
−744
−2992
−697
−2936
−2152
1923
−1974
−377
−1331
−1030
−1284
−2506
−3125
−2308
298


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


293(V)
−1738
−1298
−4281
−3921
−1737
−3979
−3665
1917
−3774
−601
−528
−3671
−3834
−3628
−3843
−3293
−1735
3205
−3215
−2770
299


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


294(E)
−833
−2344
1092
2412
−2643
−1464
−386
−2413
−146
−2369
−1505
−96
562
29
−717
−666
862
−1966
−2562
−1818
300


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


295(W)
−1380
−1116
−3614
−3026
1322
−2981
−1582
1966
−2661
1775
556
−2562
−2865
−2117
−2424
−2098
−1302
−187
2908
−629
301


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


296(T)
−350
−973
−2204
−2178
−2893
−1236
−2035
−2561
−2117
−2862
−2043
−1536
−1946
−1916
−2214
1618
3198
−1758
−3137
−2831
302


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


297(L)
−1443
−1269
−3144
−2576
−528
−3014
−1816
1945
−2155
2102
508
−2422
−2899
1193
−2133
−2129
−1369
−50
−1616
−1384
303


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


298(D)
−1826
−3682
3559
1199
−3883
−1662
−1073
−3846
−1391
−3720
−3110
−272
−2222
−760
−2283
−1471
−1913
−3321
−3864
−2864
304


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


299(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
305


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


300(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
306


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


301(Q)
−1048
−2608
205
2170
−2893
−1535
−505
−2680
−255
−2604
−1769
1814
−1849
2272
−789
−848
−1028
−2228
−2770
−2013
307


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


302(R)
1083
−1687
691
135
−2058
−1406
−178
−1755
214
−1793
−924
−145
−1553
247
1670
−383
1217
−1367
−2031
−1404
308


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


303(I)
−1915
−1536
−4077
−3667
2027
−3678
−2155
3137
−3381
144
94
−3225
−3506
−2848
−3202
−2914
−1871
345
−1522
−791
309


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


304(R)
−689
−2015
−494
24
−2395
−1582
−184
−2087
444
−2020
−1151
1161
−1687
1832
2131
626
−614
−1684
−2156
−1573
310


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


305(D)
387
−1967
1600
1359
−2275
−1391
1561
−2025
282
−1976
−1067
−25
−1525
342
1024
−369
−443
−1584
−2152
−1462
311


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


306(R)
−1460
−2315
−1793
−887
−2832
−2237
−431
−2288
2193
−2199
−1473
−946
−2245
−20
2706
−1394
−1275
591
−2248
−1961
312


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


307(V)
−941
−1027
−3099
−2832
−1692
−2234
−2324
470
−2565
−1003
−695
−2305
−2663
−2399
−2587
−1527
1858
2876
−2536
−2152
313


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


308(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
314


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


309(V)
−1090
−1215
−2097
−1824
−819
−2221
2699
−287
−1392
−1027
−591
−1674
−2482
−1446
−1482
−1482
−1143
2879
−1420
−707
315


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


310(L)
−2439
−1972
−4702
−4181
−588
−4401
−3258
1582
−3881
2757
587
−4061
−3862
−3093
−3590
−3689
−2344
−130
−2230
−2217
316


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


311(C)
2157
4166
−3012
−2973
−2780
1022
−2337
−2398
−2724
−2744
−1930
−1786
−1943
−2372
−2623
−540
−692
−1624
−3091
−2881
317


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


312(D)
−1732
−3453
3468
99
−3733
−1645
−1066
−3747
−1356
−3641
−3008
1690
−2201
−755
−2209
−1416
−1833
−3208
−3752
−2776
318


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


313(L)
−2477
−2023
−4713
−4122
1592
−4329
−2920
72
−3835
2593
2472
−3948
−3754
−2914
−3466
−3550
−2350
−634
−1927
−1830
319


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


314(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
320


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


315(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
321


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


316(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
322


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


317(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
323


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


318(K)
2
−2257
−1073
−374
−2740
−1908
−278
−2339
2328
−2192
−1373
−562
−1953
2273
1344
−952
−933
−1980
−2234
−1799
324


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


319(Y)
−3482
−2868
−3701
−3919
238
−3552
−1112
−3000
−3638
−2516
−2526
−3027
−3772
−3101
−3341
−3418
−3527
−3071
−441
4711
325


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


320(M)
−1559
−1267
−3829
−3380
−1103
−3357
−2655
805
−3067
−64
3046
−3065
−3326
−2779
−3011
−2591
−1556
2855
−2312
−1998
326


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


321(M)
1225
−469
−2256
−1679
1656
−1926
−870
90
−1396
−210
2763
−1424
−2028
−1129
−1411
−1008
712
154
−951
−586
327


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


322(T)
−738
−2094
−84
1704
−2416
−1495
−317
−2135
61
−2127
−1275
−163
−1704
1857
−405
−613
1930
−1734
−2331
−1668
328


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


323(D)
−1746
−3458
3540
90
−3744
−1650
−1081
−3767
−1381
−3662
−3036
1386
−2211
−772
−2239
−1429
−1850
−3226
−3765
−2789
329


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


324(L)
−2451
−1983
−4707
−4186
−582
−4409
−3259
1510
−3884
2778
592
−4069
−3865
−3091
−3590
−3698
−2355
−150
−2226
−2214
330


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


325(H)
−2923
−2573
−2959
−2926
826
−3449
4553
−2508
−2463
−2054
−1948
−2279
−3499
−2191
−2397
−2761
−2855
−2540
123
2920
331


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


326(K)
373
−1957
−342
1025
−2297
−1472
−98
−2018
2111
−1954
−1056
906
−1570
352
685
−424
−473
−1592
−2105
−1469
332


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


327(V)
1739
−1008
−3509
−3043
−1376
−3028
−2406
1765
−2807
−615
−334
−2718
−3093
−2585
−2823
−2226
−1263
2376
−2322
−1931
333


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


328(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
334


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


329(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
335


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


330(I)
−1758
−1302
−4331
−3970
−1756
−4054
−3748
2976
−3840
−603
−533
−3731
−3877
−3693
−3914
−3372
−1750
2505
−3265
−2824
336


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


331(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
337


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


332(Q)
1795
−1440
−730
−492
−2453
682
−812
−2151
−508
−2256
−1426
−624
−1796
2666
−901
−590
−689
−1636
−2510
−1971
338


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


333(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
339


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


334(M)
−2355
−1988
−4343
−3834
−504
−4051
−2868
105
−3385
1451
4460
−3680
−3671
−2806
−3171
−3327
−2274
−474
−2039
−1925
340


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


335(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
341


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


336(Y)
−1187
−974
−3186
−2638
−117
−2732
−1255
1905
−2270
73
1977
−2217
−2699
−1882
−2144
−1841
−1124
71
−907
3254
342


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


337(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
343


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


338(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
344


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


339(K)
−864
−1785
−860
−366
−2128
−1763
−407
−1612
2624
−1800
−1045
629
−1900
−28
62
−851
−805
1127
−2064
−1581
345


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


340(N)
602
−1686
−275
1008
−1926
−1415
1528
−1618
244
−1673
−815
1897
−1530
299
−244
−371
−391
322
−1934
−1306
346


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


341(G)
−1709
−2639
1362
−690
−3785
3257
−1671
−3805
−1946
−3792
−3137
−980
−2480
−1424
−2576
−1630
−1936
−3150
−3628
−3155
347


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


342(F)
−942
−799
−2828
−2226
1797
−2476
−1269
1109
581
1793
516
−1952
−2453
−1557
−1815
−1558
−875
52
−1138
−794
348


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


343(L)
−2451
−1983
−4707
−4186
−582
−4409
−3259
1510
−3884
2778
592
−4069
−3865
−3091
−3590
−3698
−2355
−150
−2226
−2214
349


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


344(H)
−3205
−3079
−2723
−2890
−2110
−3046
5295
−4135
−2617
−3813
−3561
−2886
−3482
−2833
−2620
−3291
−3356
−3895
−2397
−1681
350


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


345(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
351


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


346(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
352


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


347(C)
774
4452
−2162
−1688
−1962
−1478
−1302
−1474
−944
−1796
−1088
−1351
−1979
−1147
1684
−732
−719
−1116
−2225
−1881
353


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


348(L)
−2387
−1922
−4674
−4155
−617
−4366
−3250
1889
−3865
2650
558
−4023
−3847
−3098
−3586
−3647
−2296
−38
−2247
−2224
354


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


349(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
355


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


350(C)
−1489
2972
−4007
−3563
−1524
−3541
−2939
2612
−3350
−617
−413
−3224
−3470
−3129
−3335
−2770
−1475
2269
−2657
−2248
356


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


351(T)
−364
−979
−2232
−2250
−2904
−1245
−2090
−2559
−2191
−2881
−2075
−1571
−1964
−1991
−2260
905
3428
−1762
−3159
−2858
357


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


352(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
358


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


353(K)
−1716
−2632
−2004
−1008
−3336
−2379
−444
−2764
2775
−2484
−1756
−1035
−2357
2151
1811
−1592
−1477
−2481
−2391
−2172
359


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


354(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
360


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


355(V)
−1771
−1339
−4275
−3816
−1235
−3919
−3194
2139
−3617
1520
−66
−3558
−3681
−3244
−3547
−3164
−1733
2390
−2634
−2369
361


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


356(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
362


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


357(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
363


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


358(N)
−823
−1917
−96
1188
−2187
−1547
−506
−1711
−265
−1955
−1191
2711
−1815
−144
−747
−757
−815
1140
−2297
−1666
364


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


359(L)
−2153
−1779
−4360
−3884
−675
−3965
−3012
392
−3561
2726
467
−3673
−3662
−2955
−3355
−3239
−2102
1281
−2207
−2099
365


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


360(E)
1136
−2084
−175
2027
−2436
−1510
−274
−2147
1525
−2118
−1254
−175
−1692
152
−251
−593
−670
−1736
−2296
−1650
366


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


361(H)
893
−1761
1357
214
−2092
−1387
1862
−1810
229
−1825
−942
−83
−1527
293
−273
640
793
−1409
−2050
−1397
367


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


362(I)
608
−458
−2776
−2176
1666
−2202
−1113
1712
−1836
−222
338
−1782
−2245
−1512
−1731
−1292
867
1366
−1036
−684
368


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


363(P)
−922
−1912
1681
−141
−2123
−1604
−687
−1787
−550
187
−1245
−427
2677
−363
−1049
−882
−947
−1524
−2338
−1711
369


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


364(D)
−1692
−3605
3364
1256
−3770
−1599
−957
−3700
−1216
−3569
−2909
1025
−2138
−628
−2083
−1346
−1761
−3174
−3765
−2738
370


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


365(Q)
−877
−1646
−633
499
−1610
−1781
−505
−1210
−63
1648
−649
−558
−1931
2241
−360
−907
−814
−1097
−1882
−1385
371


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


366(P)
−648
−2019
1139
203
−2354
−1436
−285
−2089
29
−2086
−1217
−114
1965
1445
−492
−529
1244
−1672
−2300
−1616
372


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−571
−7108
−1646
−894
−1115
−701
−1378
*
*


367(R)
−422
−1009
−851
−304
1406
−1496
−183
−740
147
−894
−230
−440
775
21
2009
−539
−381
−568
−1136
−521
373


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−23
−6560
−7602
−894
−1115
−341
2249
*
*


368(D)
1472
−1668
1835
−70
−2356
−1385
−511
−2062
−246
−2128
−1275
−318
1353
−118
−746
−526
425
−1602
−2380
−1752
374


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


369(G)
−1044
−2230
2141
−100
−3222
2291
−982
−3045
−1033
−3050
−2258
−395
−1985
−644
−1669
858
−1207
−2428
−3250
−2493
375


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


370(Q)
−2562
−2904
−1886
−1971
−3251
−2661
−2079
−3690
−1565
−3469
−3081
−2107
−3091
4371
−1665
−2585
−2674
−3411
−3077
−2821
376


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


371(D)
−1275
−2955
2862
1330
−3205
−1556
−670
−3029
1509
−2936
−2141
−158
−1955
−290
−1213
−1025
−1281
−2554
−3111
−2272
377


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


372(V)
−1738
−1298
−4281
−3921
−1737
−3979
−3665
1917
−3774
−601
−528
−3671
−3834
−3628
−3843
−3293
−1735
3205
−3215
−2770
378


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


373(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
379


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


374(M)
−584
−1354
−847
−246
−1467
−1659
2505
−1087
212
−374
2571
−449
−1729
1171
1074
−634
−507
−876
−1617
−1128
380


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


375(P)
−910
−2031
−73
1195
−2792
−1488
−794
−2539
−629
−2588
−1788
−401
3005
−439
−1131
612
−1014
−2050
−2815
−2151
381


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


376(W)
−1588
−1300
−3783
−3197
−329
−3245
−1926
2071
−2827
1901
558
−2822
−3072
−2297
−2616
−2381
−1508
−111
3483
−1042
382


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


377(E)
−1024
−2640
1844
2310
−2908
−1498
−505
−2711
−344
−2636
−1791
−107
−1824
1521
−957
207
−1011
−2243
−2817
−2021
383


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


378(N)
−826
−2349
1089
227
−2651
−1487
−341
−2416
1494
−2346
−1475
2601
−1724
1005
−522
−657
−787
−1968
−2511
−1791
384


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


379(P)
1932
−1116
−2232
−2301
−3058
−1358
−2206
−2706
−2336
−3009
−2238
−1674
3274
−2114
−2406
−739
−914
−1913
−3260
−3019
385


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


380(V)
−914
−773
−2713
−2129
−712
−2505
−1388
1452
1084
1324
204
−1926
−2507
−1580
−1808
−1591
−859
1713
−1424
−1081
386


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


381(Y)
−1484
−2331
−1762
−887
−2436
−2254
−420
−2325
2137
−2195
−1475
−949
−2258
−39
1983
−1411
−1295
−2075
−2087
2868
387


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


382(E)
1256
−1890
−206
1353
−2196
−1401
−89
−1930
812
−1898
−996
−45
547
1252
−162
−356
−414
−1507
−2083
−1416
388


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


383(Q)
−752
−2272
1586
1407
−2561
−1448
−308
−2329
−23
−2276
−1396
−71
−1677
1749
−577
−590
1569
−1881
−2459
−1727
389


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


384(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
390


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


385(H)
−964
−2089
−200
−136
−2264
−1600
3833
−2320
−296
−2338
−1558
1362
1479
−276
−699
−881
−992
−1924
−2364
−1652
391


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


386(L)
−2451
−1983
−4707
−4186
−582
−4409
−3259
1510
−3884
2778
592
−4069
−3865
−3091
−3590
−3698
−2355
−150
−2226
−2214
392


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


387(Q)
1643
−1017
−1196
−721
−1189
−1714
−668
1336
−497
−907
−297
−823
−1893
2044
−794
−784
−569
−339
−1579
−1135
393


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


388(I)
−1760
−1308
−4323
−3961
−1730
−4039
−3721
3156
−3825
−575
−512
−3720
−3867
−3669
−3893
−3356
−1753
2241
−3236
−2802
394


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


389(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
395


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


390(K)
−1259
−2115
−1267
−676
−970
−2105
1794
−2040
2549
−1955
−1282
−808
−2165
−167
114
−1192
−1140
−1801
−1301
2517
396


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


391(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
397


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


392(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
398


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


393(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
399


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


394(A)
3121
−934
−2489
−2561
−3081
−1203
−2295
−2766
−2533
−3080
−2234
−1669
−1953
−2234
−2533
936
−746
−1844
−3331
−3090
400


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


395(E)
−522
−1773
−240
1676
−2248
−1396
−289
−1968
50
−1989
−1115
−174
1198
131
−448
1226
677
−1538
−2214
−1565
401


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


396(E)
−1481
−3230
1425
2936
−3481
751
−843
−3354
−954
−3256
−2520
−187
−2057
−492
−1711
−1193
−1527
−2852
−3445
−2523
402


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


397(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
403


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


398(A)
2847
−932
−2454
−2477
−3066
−1198
−2236
−2763
−2439
−3057
−2202
−1635
−1940
−2152
−2471
1777
−731
−1840
−3306
−3056
404


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


399(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
405


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


400(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
406


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


401(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
407


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


402(I)
−1761
−1312
−4317
−3954
−1713
−4027
−3703
3225
−3814
−556
−498
−3712
−3859
−3653
−3877
−3344
−1754
2110
−3216
−2787
408


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


403(S)
−348
−981
−2200
−2194
−2989
−1227
−2073
−2686
−2157
−2970
−2136
−1541
−1946
−1946
−2253
3060
1398
−1824
−3217
−2916
409


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


404(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
410


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


405(V)
−917
−809
−2556
−1976
−827
−2491
−1367
1339
1455
721
94
−1841
−2501
−1487
−1710
−1570
−863
2038
−1514
−1151
411


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


406(K)
−1386
−2643
−447
1824
−3108
−1893
−570
−2762
2860
−2616
−1848
−552
−2117
−166
−3
−1217
−1300
−2388
−2647
−2154
412


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


407(N)
−537
−1563
−449
−36
−1889
1143
−307
−1529
932
−1655
−844
1794
−1658
73
−356
−518
−516
924
−1962
−1392
413


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


408(P)
−894
−2181
−369
1705
−2576
−1650
−357
−2268
243
−2210
−1375
−330
2093
63
1619
−774
−835
−1876
−2347
−1769
414


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


409(V)
−419
−634
−1376
−807
1053
−1737
−499
−198
−623
−505
178
600
−1807
−475
475
313
−360
1389
−1016
1303
415


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


410(I)
−1282
−1082
−3022
−2555
2426
−2683
1767
2555
−2191
−443
−88
−2038
−2692
−1794
−2075
−1793
−1220
−317
−361
552
416


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


411(T)
−499
−1595
−431
966
−1830
−1487
−185
−1449
1092
−1574
−754
−207
−1601
213
−206
−458
2067
159
−1877
−1296
417


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


412(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
418


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


413(P)
−632
−1230
−2074
−2144
−2996
−1453
−2116
−2631
−2128
−2928
−2213
−1658
3610
−2006
−2221
−852
1302
−1931
−3185
−2917
419


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


414(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
420


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


415(R)
−1454
−2316
−1780
−878
−2834
−2232
−428
−2292
2281
−2200
−1473
−940
−2240
−17
2627
−1386
−1270
588
−2249
−1960
421


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


416(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
422


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


417(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
423


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


418(D)
−1572
−3426
2573
2447
−3613
−1583
−879
−3513
−1050
−3393
−2684
1292
−2085
−535
−1855
−1253
−1623
−3000
−3585
−2609
424


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


419(S)
−879
−1989
1498
−177
−3045
1600
−939
−2843
−904
−2867
−2046
−438
−1922
−591
−1483
2171
−1044
−2226
−3072
−2372
425


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


420(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
426


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


421(Q)
−705
−1925
−199
2112
917
−1534
−288
−1824
42
−1842
−1054
−210
−1709
2163
−420
−611
−656
−1502
−1997
−1291
427


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


422(H)
−569
−2048
1450
1526
−2349
−1405
1830
−2103
181
−2058
−1157
−37
−1569
272
−349
713
620
−1662
−2240
−1537
428


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


423(C)
1626
2878
−2671
−2107
1264
−1968
−1091
233
−1777
−334
250
−1672
−2128
−1459
−1691
−1096
−529
1209
−1066
−704
429


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


424(M)
−2042
−1634
−4379
−3826
−659
−3976
−2899
2765
−3546
1204
3085
−3605
−3604
−2896
−3318
−3183
−1961
195
−2135
−2058
430


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


425(E)
412
−2447
1356
2379
−2747
−1477
−445
−2527
−243
−2477
−1622
−107
855
−36
−831
−730
−894
−2073
−2668
−1906
431


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


426(A)
2822
−1031
−2418
−2539
−3226
1898
−2364
−2941
−2626
−3229
−2379
−1722
−2026
−2302
−2634
−654
−848
−1983
−3415
−3226
432


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


427(I)
−1772
−1325
−4307
−3877
−1405
−3993
−3383
2935
−3705
820
−217
−3632
−3761
−3400
−3682
−3260
−1742
2033
−2838
−2525
433


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


428(L)
−875
−1634
−575
959
−1581
−1769
−525
−1179
−135
1884
−625
−547
−1931
1405
−450
−909
−816
−1074
−1883
−1383
434


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


429(A)
1705
−1826
−180
949
−2318
−1410
−359
−2041
−53
−2067
−1204
1001
−1652
52
−561
1232
−595
−1609
−2298
−1643
435


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


430(D)
−1074
−2458
2381
60
−2921
1927
−658
−2710
−463
−2675
−1860
−271
−1918
−276
866
−915
−1100
−2245
−2845
−2124
436


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


431(K)
−688
−2117
785
888
−2469
−1529
−187
−2189
2380
−2106
−1221
−162
−1661
256
1134
−553
−619
−1760
−2240
−1607
437


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


432(I)
−2019
−1582
−4380
−3941
−1000
−4086
−3253
3295
−3671
1100
145
−3736
−3783
−3222
−3556
−3378
−1976
657
−2517
−2289
438


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


433(Q)
−490
−1797
−369
171
−2078
−1457
1762
−1779
1157
−1780
−905
1165
−1550
1798
−48
−396
−422
725
−1986
−1366
439


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


434(A)
1954
−1836
1733
−180
−2714
−1429
−806
−2438
−679
−2518
−1698
−430
1775
−448
−1211
−736
−894
−1923
−2765
−2117
440


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


435(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
441


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


436(D)
−1736
−3455
3490
97
−3737
−1646
−1070
−3753
−1363
−3647
−3016
1602
−2204
−760
−2218
−1420
−1838
−3213
−3756
−2780
442


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


437(V)
−1721
−1302
−4229
−3874
−1705
−3894
−3582
1607
−3706
−582
−513
−3610
−3786
−3559
−3767
−3209
−1725
3294
−3158
−2712
443


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


438(V)
594
−988
−3391
−2911
−1164
−2888
−2187
845
−2637
765
−154
−2576
−2962
−2387
−2622
−2074
−1205
2800
−2084
−1724
444


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


439(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
445


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


440(I)
−1754
−1308
−4295
−3867
−1434
−3978
−3377
2661
−3697
862
−247
−3617
−3754
−3406
−3679
−3243
−1725
2373
−2852
−2526
446


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


441(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
447


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


442(Y)
−1321
−1438
−1994
−1608
2186
527
−450
−1117
−1481
−1211
−693
1178
−2522
−1217
−1665
−1518
−1275
−1021
−198
3178
448


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


443(C)
−675
2205
−2544
972
−572
−2236
−1121
1373
−1671
679
261
−1700
−2270
−1403
−1668
−1311
−621
1601
−1150
−790
449


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


444(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
450


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


445(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
451


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


446(K)
−1060
−2058
−1088
−460
−2432
−1917
−357
−1970
2801
−1978
−1220
−632
−1990
1339
367
−999
−946
536
−2145
−1717
452


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


447(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
453


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


448(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
454


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


449(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
455


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


450(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
456


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


451(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
457


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


452(P)
−1659
−2241
−2022
−1646
−3185
−2242
−1373
−3000
−450
−2936
−2274
−1624
3435
−1065
2095
−1730
−1750
−2593
−2816
−2613
458


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


453(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
459


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


454(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
460


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


455(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3965
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
461


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


456(K)
1368
−1491
−763
−332
−2319
−1417
−551
−1998
1786
−2068
−1221
−500
−1721
−160
−470
1631
−587
−1532
−2299
−1754
462


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


457(P)
−1500
−1738
−2514
−2380
−1555
−2358
−2022
−1126
−2063
1224
−841
−2189
3436
−2061
−2129
−1822
−1674
−1231
−2290
−1878
463


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


458(T)
−351
−974
−2208
−2185
−2894
−1237
−2041
−2561
−2125
−2863
−2046
−1539
−1948
−1923
−2218
1543
3230
−1758
−3139
−2834
464


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


459(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
465


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


460(M)
2706
−986
−2433
−2144
−1502
−1684
−1706
−700
−1858
−968
2744
−1705
−2188
−1713
−1932
−963
−862
−592
−2145
−1794
466


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


461(I)
−2103
−1659
−4461
−3992
−869
−4152
−3233
3082
−3723
1619
290
−3801
−3788
−3171
−3557
−3432
−2046
487
−2418
−2265
467


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


462(I)
−1761
−1312
−4317
−3954
−1713
−4027
−3703
3225
−3814
−556
−498
−3712
−3859
−3653
−3877
−3344
−1754
2110
−3216
−2787
468


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


463(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
469


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


464(K)
1641
−2033
−323
914
−2415
−1565
−296
−2097
2052
−2080
−1233
−257
−1736
125
−133
−646
−702
−1707
−2258
−1657
470


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


465(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
471


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


466(L)
−1699
−1807
−2268
−1925
−830
−2795
−1551
−455
−1225
2510
90
−1958
−2845
1927
−1308
−2067
−1651
−846
−1841
−1454
472


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


467(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
473


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


468(D)
−853
−2415
2115
1717
−2702
−1468
−378
−2484
1085
−2417
−1546
−84
−1732
41
−699
696
−824
−2025
−2594
−1839
474


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


469(S)
−892
−1780
−931
−688
−2757
−1643
−830
−2472
1671
−2492
−1708
−799
−2018
−468
−365
2676
−1004
−1981
−2598
−2130
475


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


470(C)
−1135
3503
−3700
−3406
−1670
−2549
−2675
653
−3101
−916
−667
−2727
−2925
−2870
−3030
−1868
−1288
2927
−2619
−2222
476


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


471(A)
2590
−1035
−2404
−2530
−3236
2290
−2365
−2954
−2627
−3240
−2389
−1719
−2027
−2302
−2637
−656
−851
−1991
−3423
−3234
477


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


472(L)
−2632
−2152
−4630
−4185
1767
−4324
−2442
−61
−3879
2789
563
−3833
−3823
−2970
−3513
−3609
−2518
−738
−1527
−945
478


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


473(I)
−2073
−1632
−4434
−3975
−911
−4130
−3238
3164
−3706
1451
244
−3779
−3785
−3187
−3557
−3413
−2021
546
−2449
−2273
479


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


474(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
480


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


475(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
481


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


476(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
482


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


477(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
483


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


478(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
484


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


479(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
485


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


480(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
486


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


481(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
487


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


482(T)
−359
−976
−2225
−2229
−2900
−1242
−2074
−2560
−2170
−2875
−2064
−1561
−1958
−1969
−2247
1110
3375
−1760
−3152
−2850
488


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


483(Y)
−3402
−2632
−3941
−4011
1064
−3924
3388
−2526
−3541
−1996
−1973
−2625
−3821
−2664
−3170
−3135
−3280
−2619
3420
3756
489


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


484(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
490


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


485(M)
−2322
−1904
−4536
−3951
2387
−4112
−2676
67
−3649
2034
3156
−3710
−3633
−2803
−3311
−3309
−2204
−588
−1794
−1586
491


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


486(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
492


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


487(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
493


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


488(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
494


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


489(H)
−3205
−3079
−2723
−2890
−2110
−3046
5295
−4135
−2617
−3813
−3561
−2886
−3482
−2833
−2620
−3291
−3356
−3895
−2397
−1681
495


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


490(V)
−1754
−1297
−4329
−3968
−1770
−4053
−3752
2604
−3840
−621
−545
−3728
−3878
−3699
−3917
−3370
−1746
2859
−3276
−2829
496


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


491(A)
2587
−828
−2477
−2155
−1837
−1468
−1728
−743
−1941
−1564
−954
−1607
−2033
−1725
−2034
−738
1178
1108
−2310
−1972
497


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


492(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
498


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


493(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
499


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


494(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
500


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


495(Y)
−866
−976
−1863
−1331
1353
−2145
1318
−556
−1116
−777
−173
−1242
−2197
1714
−1301
−1173
−802
888
−445
2749
501


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


496(D)
417
−1831
1647
1094
−2065
−1488
−353
−1618
−107
−1820
−1019
−189
−1698
30
−623
−603
−643
1629
−2154
−1520
502


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


497(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
503


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


498(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
504


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


499(T)
492
−1190
−706
−181
−1475
311
−333
−1099
−81
71
−509
570
1113
−6
−509
−450
1123
−835
−1680
−1161
505


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


500(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
506


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


501(A)
3103
−1036
−2445
−2572
−3222
1051
−2380
−2930
−2650
−3226
−2381
−1739
−2034
−2327
−2648
−664
−857
−1981
−3412
−3228
507


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


502(L)
−2239
−1892
−3711
−3400
301
−3520
−1210
−542
−2948
2564
−35
−2786
−3395
−2438
−2750
−2747
−2165
−945
−573
2562
508


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


503(V)
−1757
−1387
−4101
−3681
−1174
−3714
−3031
880
−3410
1254
−60
−3407
−3585
−3094
−3354
−2984
−1743
3014
−2536
−2219
509


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


504(Q)
−982
−2251
−866
971
−2711
−1822
−252
−2340
1444
−2194
−1356
−464
−1885
2646
1632
−858
−863
−1958
−2245
−1765
510


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


505(E)
−1162
−2771
2137
2239
−3046
−1526
−626
−2849
−546
−2792
−1983
−145
−1905
−242
−1192
−940
1396
−2385
−2990
−2169
511


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


506(G)
−1707
−2684
1591
−614
−3783
3190
−1613
−3795
−1887
−3775
−3119
−915
−2456
−1358
−2539
−1610
−1924
−3150
−3636
−3124
512


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


507(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
513


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


508(M)
−473
−522
−1819
−1236
−468
−1879
−687
1519
−996
566
1677
−1154
−1937
836
−1131
1079
−413
102
−957
−585
514


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


509(I)
−1761
−1312
−4317
−3954
−1713
−4027
−3703
3225
−3814
−556
−498
−3712
−3859
−3653
−3877
−3344
−1754
2110
−3216
−2787
515


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


510(T)
782
−1467
−550
1029
−2202
−1425
−709
−1791
−472
−1993
−1203
−528
−1787
−368
−902
−617
2685
−1400
−2333
−1783
516


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


511(I)
−1766
−1333
−4283
−3923
−1635
−3967
−3619
3388
−3759
−473
−437
−3672
−3822
−3576
−3804
−3285
−1764
1695
−3126
−2717
517


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


512(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
518


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


513(A)
2705
−1451
−1036
−913
−2506
−1504
−1143
−2174
−794
−2337
−1613
−946
−1993
2040
−1061
−809
−910
−1703
−2633
−2156
519


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


514(H)
−615
−1680
1444
66
−1883
168
2650
−1558
−86
−1691
−891
−223
−1680
31
−577
−571
−585
1267
−2007
−1397
520


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


515(K)
−654
−2006
−546
42
−2376
−1581
−133
−2066
1935
−1987
−1107
1132
−1658
1043
1058
−540
1180
−1660
−2113
−1532
521


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


516(N)
−933
−2085
−946
−284
−2472
−1822
−253
−2090
1711
76
−1204
1918
−1876
175
1799
−841
−817
−1755
−2132
−1663
522


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


517(E)
−416
−987
−843
1107
−1070
−1583
−338
−623
−183
879
−172
−489
−1679
−94
−565
544
813
265
−1379
−905
523


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


518(I)
−2258
−1804
−4588
−4084
−706
−4269
−3231
2527
−3807
2292
465
−3923
−3814
−3118
−3570
−3544
−2181
190
−2303
−2237
524


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


519(Q)
−477
−1909
958
282
−2211
−1389
1484
−1953
285
−1921
−1018
−32
−1517
2318
−225
630
559
−1525
−2110
−1430
525


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


520(L)
−2127
−1743
−4402
−3796
1257
−3918
−2674
149
−3492
2527
2164
−3553
−3509
−2714
−3181
−3095
−2019
570
−1870
−1818
526


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


521(N)
−723
−2217
958
236
−2518
−1466
1611
−2279
1719
−2217
−1334
2285
−1666
166
−401
−570
−677
−1837
−2382
−1678
527


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


522(V)
−1754
−1297
−4330
−3968
−1770
−4053
−3752
2623
−3841
−620
−545
−3729
−3878
−3699
−3918
−3371
−1746
2846
−3277
−2830
528


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


523(S)
1545
−974
−2003
−1825
−2867
−1206
−1790
−2580
−1788
−2795
−1932
−1362
1826
−1586
−1999
2362
−672
−1755
−3057
−2721
529


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


524(D)
−1776
−3649
3326
1869
−3838
−1642
−1031
−3788
−1322
−3660
−3029
−245
−2192
−711
−2201
−1425
−1855
−3264
−3821
−2816
530


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


525(E)
423
−2950
1944
2696
−3223
−1545
−718
−3047
−715
−2979
−2196
−161
−1968
−347
−1403
−1043
−1314
−2569
−3177
−2316
531


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


526(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
532


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


527(L)
−2339
−1899
−4618
−4042
1570
−4204
−2849
1440
−3758
2558
676
−3825
−3700
−2902
−3418
−3418
−2226
−382
−1924
−1778
533


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


528(A)
2338
−1990
−241
938
−2395
−1557
−423
−2061
954
−2103
−1286
−301
−1791
−26
−375
−717
−784
−1691
−2330
−1728
534


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


529(R)
524
−2098
−789
−146
−2504
−1729
1632
−2153
1229
−2054
−1204
−379
−1789
1328
2313
−719
−724
−1774
−2150
−1637
535


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


530(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
536


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


531(R)
−1895
−2713
−2327
−1192
−3484
−2502
−481
−2856
2144
−2544
−1842
−1161
−2458
1393
3023
−1770
−1619
−2599
−2421
−2259
537


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


532(A)
2935
−1714
−553
857
−2769
−1546
−1218
−2333
−1106
−2591
−1873
−809
−2065
−934
−1502
−954
−1103
−1872
−2898
−2374
538


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


533(A)
1291
−1874
−176
1227
−2177
−1392
−109
−1909
277
−1891
−995
1134
−1522
1248
−228
−361
562
−1492
−2090
−1419
539


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


534(W)
−805
−687
−2581
−2028
138
−2236
−697
897
−1681
−421
141
−1645
−2282
−1369
−1627
−1315
636
−90
4479
1809
540


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


535(H)
−408
−1801
−274
1284
−2096
−1385
1500
−1822
1168
−1802
−899
−33
−1479
1381
−102
−303
595
221
−1996
−1339
541


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


536(Q)
−650
−1737
−627
−72
−1981
−1615
−209
−1625
1223
392
−866
−318
1222
2120
50
−598
−572
−1326
−1932
−1394
542


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


537(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
543


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−324
−7108
−2368
−894
−1115
−701
−1378
*
*


538(A)
2195
−924
−968
−546
−1397
−1356
−583
−812
−365
−1167
−487
−618
−1660
1324
−684
−483
−404
462
−1703
−1242
544


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−19
−6804
−7846
−894
−1115
−428
−1961
*
*


539(P)
411
−1017
−1886
−1616
−1600
−1588
−1411
−962
−1408
495
−755
−1384
3156
−1323
−1577
−847
−785
−783
−2111
−1716
545


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


540(R)
−1612
−2397
−2037
−1033
−2897
−2352
−458
−2365
2184
665
−1520
−1051
−2334
−51
2602
−1545
−1395
−2143
−2262
−2014
546


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


541(Y)
712
−796
−2334
−1883
−370
−2028
−986
−143
−1607
−663
−131
−1587
−2243
−1383
−1656
−1178
−771
1114
−965
3479
547


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


542(T)
−527
−1669
1091
−27
−2315
−1379
−443
−2033
−151
−2081
−1218
−282
557
−41
−650
1128
2077
−1576
−2321
−1690
548


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


543(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
549


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


544(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
550


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


545(V)
−1747
−1296
−4310
−3948
−1758
−4023
−3716
2215
−3813
−615
−540
−3705
−3860
−3670
−3887
−3339
−1741
3087
−3252
−2806
551


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


546(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
552


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


547(A)
2404
−890
−1926
−1629
−1803
1275
−1415
−1282
−1490
392
−963
−1316
−1930
−1328
−1674
−654
−644
−952
−2187
−1810
553


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


548(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
554


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


549(Y)
−3621
−2707
−4176
−4424
2950
−4049
−394
−2539
−4002
−1942
−1987
−2749
−3933
−2854
−3451
−3299
−3499
−2690
349
4094
555


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


550(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
556


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


551(H)
−1741
−2627
−2070
−1046
−3303
−2401
2713
−2751
2478
−2476
−1755
−1061
−2375
−27
2379
−1621
−1497
−2477
−2379
−2161
557


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


552(L)
−1014
−876
−2956
−2408
−582
−2550
−1529
1721
−2079
2042
345
−2114
−2581
−1775
−2028
454
−980
286
−1414
−1096
558


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


553(V)
933
−842
−2818
−2467
−1542
−1870
−1890
154
−2226
−1095
−617
−1932
−2326
−1995
−2259
−1126
1070
2769
−2180
−1826
559


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


554(S)
−787
−1522
−1486
−1172
−2714
−1599
−1112
−2500
−433
−2563
−1791
−1110
−2067
−796
1351
2916
−989
−1943
−2648
−2234
560


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


555(S)
−326
−1010
−1779
−1541
−2691
−1234
−1566
−2386
−1486
−2594
−1749
−1228
1196
−1330
−1747
2396
1967
−1662
−2876
−2496
561


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


556(A)
3121
−934
−2489
−2561
−3081
−1203
−2295
−2766
−2533
−3080
−2234
−1669
−1953
−2234
−2533
936
−746
−1844
−3331
−3090
562


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


557(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
563


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


558(R)
−586
−1873
−516
979
−2188
−1543
−123
−1869
1290
−353
−980
−202
−1622
314
1886
−491
782
−1495
−2024
−1439
564


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


559(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
565


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


560(C)
2804
3772
−3185
−3198
−2739
−1303
−2462
−2065
−2882
−2628
−1924
−1927
−2044
−2547
−2727
−661
−799
−1463
−3099
−2886
566


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


561(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
567


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


562(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
568


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*


563(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
569


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249


—
−21
−6715
−7757
−894
−1115
−701
−1378
*
*


564(F)
−525
−445
−2202
−1627
1946
−2001
−744
1247
−1346
952
561
1079
−2030
−1067
−1362
−1067
−465
338
−714
−230
570


—
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*


—
*
*
*
*
*
*
*
*
0











HMMER2.0 [2.2g]
Program name and version


NAME dhad_for_hmm
Name of the input sequence alignment file


LENG 564
Length of the alignment: include indels


ALPH Amino
Type of residues


MAP yes
Map of the match states to the columns of the alignment


COM/app/public/hmmer/current/bin/hmmbuild -F dhad-exp_hmm dhad_for_hmm.aln
Commands used to generate the file: this one means



that hmmbuild (default patrameters) was applied to the alignment file


COM/app/public/hmmer/current/bin/hmmcalibrate dhad-exp_hmm
Commands used to generate the file: this one means that hmmcalibrate



(default parametrs) was applied to the hmm profile


NSEQ 8
Number of sequences in the alignment file


DATE Tue Jun 3 10:48:24 2008
When was the file generated


XT −8455 −4 −1000 −1000 −8455 −4 −8455 −4


NULT −4 −8455
The transition probability distribution for the null model (single G state).


NULE 595 −1558 85 338 −294 453 −1158 197 249 902 −1085 −142 −21 −313 45 531 201 384 −199
The symbol emission probability



distribution for the null model (G state); consists of K (e.g. 4 or 20) integers. The null



probability used to convert these back to model probabilities is 1/K.


EVD −499.650970 0.086142
The extreme value distribution parameters μ and lambda respectively; both floating point values.



Lambda is positive and nonzero.



These values are set when the model is calibrated with hmmcalibrate.

## Claims

1. A recombinant yeast cell comprising a heterologous gene encoding a mitochondrial localized polypeptide having α-keto acid decarboxylase activity and wherein the mitochondria is substantially devoid of a functional polypeptide having threonine deaminase activity and a functional polypeptide having isopropylmalate synthase activity.

2. The yeast cell of claim 1 wherein the cell produces isobutanol in the mitochondria.

3. The yeast cell of claim 2 wherein the cell further comprises genes encoding mitochondrial localized polypeptides having ketol-acid reductoisomerase activity and dihydroxy-acid dehydratase activity which are overexpressed.

4. The yeast cell of claim 3 wherein the mitochondria is further substantially devoid of a functional polypeptide having an enzyme activity selected from branched chain amino acid transaminase activity, pyruvate dehydrogenase activity, or a combination thereof.

5. The yeast cell of claim 1 selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.

6. The yeast cell of claim 1 wherein the α-keto acid decarboxylase activity is defined by the enzyme classification number EC 4.1.1.72.

7. The yeast cell of claim 4 wherein:
a) the threonine deaminase activity is defined by the enzyme classification number EC 4.3.1.19;
b) the isopropylmalate synthase activity is defined by the enzyme classification number EC 2.3.3.13;
c) the branched chain amino acid transaminase activity is defined by the enzyme classification number EC 2.6.1.42; and
d) the pyruvate dehydrogenase activity is defined by the enzyme classification number EC 1.2.4.1.

8. The yeast cell of claim 4 wherein the polypeptide having the pyruvate dehydrogenase activity is a multienzyme complex comprising proteins selected from the group consisting of: PDA1, PDA1, PDB1, LAT1, LPD1, and PDX1.

9. A method for making isobutanol comprising growing the yeast host cell of any of claim 3, and 4 under conditions whereby isobutanol is produced.