# Patent

## Title
Recombinant yeast host cell with Fe-S cluster proteins and methods of using thereof

## Abstract

Yeast strains were engineered that have increased activity of heterologous proteins that require binding of an Fe—S cluster for their activity. The yeast strains have reduced activity of an endogenous Fe—S protein. Activities of heterologous fungal or plant 2Fe-2S dihydroxy-acid dehydratases and Fe—S propanediol dehydratase reactivase were increased for increased production of products made using biosynthetic pathways including these enzymes, such as valine, isoleucine, leucine, pantothenic acid (vitamin B5), isobutanol, 2-butanone and 2-butanol.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 12/569,069, filed Sep. 29, 2009, which claims the benefit of priority to U.S. Provisional Application Nos. 61/100,801 filed Sep. 29, 2008 and 61/100,806 filed Sep. 29, 2008. The entirety of each is hereby incorporated by reference.


REFERENCE SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The content of the electronically submitted sequence listing (Name: CL4213USCNT.txt; Size: 707,401 bytes; and Date of Creation: Sep. 27, 2011) is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to the field of industrial microbiology and the expression of proteins that require an iron-sulfur cluster for activity. More specifically, expression of heterologous Fe—S protein activity in yeast cells is improved through specific host gene inactivation.
BACKGROUND OF THE INVENTION
Engineering of yeast for fermentative production of commercial products is an active and growing field. Enzymatic pathways engineered for biosynthesis of some products include enzymes that require binding of an iron-sulfur (Fe—S) cluster for activity. Dihydroxy-acid dehydratase (DHAD) is one example. DHAD is part of naturally occurring biosynthetic pathways producing valine, isoleucine, leucine and pantothenic acid (vitamin B5). Increased expression of DHAD activity is desired for enhanced microbial production of branched chain amino acids or of pantothenic acid. In addition, DHAD catalyzed conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is a common step in the multiple isobutanol biosynthetic pathways that are disclosed in co-pending US Patent Pub No. US 20070092957 A1. Disclosed therein is engineering of recombinant microorganisms for production of isobutanol, which is useful as a fuel additive and whose availability may reduce the demand for petrochemical fuels.
Diol dehydratase provides an enzyme activity in a biosynthetic pathway for production of 2-butanone and 2-butanol that is disclosed in co-pending US Patent Pub No. US 2007-0292927A1. Disclosed in US Patent Pub No. US20090155870 is a butanediol dehydratase that is useful for expression in this pathway due to its coenzyme B-12 independence. A diol dehydratase reactivase that is an Fe—S cluster protein required for activity of the B12-independent butanediol dehydratase, is also disclosed in US Patent Pub No. US20090155870. 2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent, extractant and activator of oxidative reactions, as well as a substrate for chemical synthesis of 2-butanol. 2-butanol is useful as a fuel additive, whose availability may reduce the demand for petrochemical fuels.
For improved production of compounds synthesized in pathways including an Fe—S cluster containing enzyme, it is desirable to provide a host cell capable of expressing high levels of this enzymatic activity in the production host of interest. Whereas a number of commercially relevant bacteria and yeast can express activity of Fe—S cluster containing proteins, this activity is at levels far below what is commercially useful for enhancing introduced biosynthetic pathways. Consequently a need exists for the discovery of host cells capable of expressing activity of Fe—S cluster containing proteins at levels high enough to enhance introduced pathways that have Fe—S requirements. Obtaining high functional expression of heterologous Fe—S cluster containing enzymes is problematic due to the Fe—S cluster requirement, which involves availability and proper loading of the cluster into the apo-protein.
SUMMARY OF THE INVENTION
Provided herein are recombinant yeast host cells comprising at least one heterologous Fe—S cluster protein wherein the yeast host has reduced expression of at least one endogenous Fe—S cluster protein.
The recombinant yeast cell may be grown under suitable conditions for the production of products including isobutanol, 2-butanol and 2-butanone.
In one aspect, the recombinant yeast cell comprises a disruption in the gene encoding the at least one endogenous Fe—S cluster protein.
In another aspect, the endogenous Fe—S cluster protein is selected from the group consisting of dihydroxy-acid dehydratase, isopropylmalate dehydratase, sulfite reductase, glutamate dehyddrogenase, biotin synthase, aconitase, homoaconitase, lipoate synthase, ferredoxin maturation, NADH ubiquinone oxidoreductase, succinate dehydrogenase, ubiquinol-cytochrome-c reductase, ABC protein Rli1 NTPase Nbp35, and hydrogenase-like protein.
In another aspect, the yeast is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia. 
In another aspect, the endogenous Fe—S protein is expressed in the mitochondria, and in another embodiment, the endogenous Fe—S cluster protein has an activity selected from the group consisting of: dihydroxy-acid dehydratase and isopropylmalate dehydratase activity.
In another aspect, the host cell is Saccharomyces expressing a gene encoding a polypeptide having the amino acid sequence as set forth in SEQ ID NO:114.
In some embodiments, the at least one heterologous Fe—S cluster protein is selected from the group consisting of fungal 2Fe-2S dihydroxy-acid dehydratases and plant 2Fe-2S dihydroxy-acid dehydratases. In one embodiment, the heterologous fungal or plant 2Fe-2S cluster dihydroxy-acid dehydratase is expressed in the cytosol. In one embodiment, the heterologous fungal or plant 2Fe-2S cluster dihydroxy-acid dehydratase is a polypeptide having an amino acid sequence that matches the Profile HMM of table 9 with an E value of <10−5 wherein the polypeptide additionally comprises all three conserved cysteines, corresponding to positions 56, 129, and 201 in the amino acids sequences of the Streptococcus mutans DHAD enzyme corresponding to SEQ ID NO:179. In one embodiment, the heterologous fungal or plant 2Fe-2S cluster dihydroxy-acid dehydratase is a polypeptide having an amino acid sequence that has at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150 and 152. In one embodiment, the heterologous fungal or plant 2Fe-2S cluster dihydroxy-acid dehydratase is a polypeptide having an amino acid sequence that is at least about 90% identical to SEQ ID NO:114 using 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.
In another aspect, a method for the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is provided, said method comprising:
a) providing (1) a recombinant yeast host cell comprising at least one heterologous gene encoding a 2Fe-2S dihydroxy-acid dehydratase wherein the recombinant yeast host cell has reduced activity of at least one endogenous Fe—S cluster protein; and (2) a source of 2,3-dihydroxyisovalerate; and
b) growing the recombinant host cell of (a) with said source of 2,3-dihydroxyisovalerate under conditions where the 2,3-dihydroxyisovalerate is converted by the host cell to α-ketoisovalerate.
In another aspect, a method for the conversion of 2,3-butanediol to 2-butanone is provided, said method comprising:
a) providing (1) a recombinant yeast host cell comprising at least one heterologus gene encoding a Fe—S propanediol dehydratase reactivase wherein the recombinant yeast host cell has reduced activity of at least one endogenous Fe—S cluster protein; and (2) a source of 2,3-butanediol; and
b) growing the recombinant host cell of (a) with said source of 2,3-butanediol under conditions where 2,3-butanediol is converted by the hots cell to 2-butanone.
Also provided is a method for the production of isobutanol comprising growing a recombinant yeast host cell disclosed herein under conditions wherein isobutanol is produced.
In other embodiments, the at least one heterologous Fe—S cluster protein has Fe—S propanediol dehydratase reactivase activity. In some embodiments, the at least one heterologous Fe—S cluster protein having Fe—S propanediol dehydratase reactivase activity is a propanediol deydratase reactivase having an amino acid sequence that is at least about 90% identical to the amino acid sequence as set forth in SEQ ID NO:44 using 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.
In some embodiments, the cell produces 2-butanol, and in some embodiments the cell produces 2-butanone. In some embodiments, the cell comprises a 2-butanol biosynthetic pathway, and in some embodiments, the cell comprises a 2-butanone biosynthetic pathway.



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 biosynthetic pathways for isobutanol production.
FIG. 2 shows a biosynthetic pathway for 2-butanone and 2-butanol production.



Table 9 is a table of the Profile HMM for dihydroxy-acid dehydratases based on enzymes with assayed function prepared as described in Example 1. Table 9 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







Inactivation target Fe—S protein encoding genes










SEQ ID NO:
SEQ ID NO:


Organism and gene
Nucleic Acid
Peptide












Saccharomyces cerevisiae LEU1
1
2


Schizosaccharomyces pombe LEU1
3
4


Candida galbrata CBS 138 LEU1
5
6


Candida albicans SC 5314 LEU1
7
8


Kluyveromyces lactis LEU1
9
10


Yarrowia lipolytica LEU1
11
12


Pichia stipitis LEU1
13
14


Saccharomyces cerevisiae YJM789
111
112


ILV3




Schizosaccharomyces pombe ILV3
93
94


Candida galbrata CBS 138 ILV3
107
108


Candida albicans SC5314 ILV3
101
102


Kluyveromyces lactis ILV3
113
114


Yarrowia lipolytica ILV3
105
106


Pichia stipitis CBS 6054 ILV3
103
104


Saccharomyces cerevisiae ACO1
153
154


Schizosaccharomyces pombe
155
156


(chromosome II) ACO1




Schizosaccharomyces pombe
157
158


(chromosome I) ACO1




Kluyveromyces lactis NRRL Y-1140
159
160


ACO1




Candida albicans SC5314 ACO1
161
162


Yarrowia lipolytica CLIB122 ACO1
163
164


Pichia stipitis CBS 6054 ACO1
165
166


Candida glabrata CBS138
167
168


(chromosome F) ACO1




Candida glabrata CBS138
169
170


(chromosome D) ACO1




Candida glabrata CBS138
171
172


(chromosome K) ACO1
















TABLE 2







Fungal and plant 2Fe—2S DHADs in addition to those in Table 1










SEQ ID NO:
SEQ ID NO:


Description
Nucleic acid
Peptide












Chlamydomonas reinhardtii
45
46


Ostreococcus lucimarinus CCE9901
47
48


Vitis vinifera
49
50


(Unnamed protein product: CAO71581.1)




Vitis vinifera (CAN67446.1)
51
52


Arabidopsis thaliana
53
54


Oryza sativa (indica cultivar-group)
55
56


Physcomitrella patens subsp. patens
57
58


Chaetomium globosum CBS 148.51
59
60


Neurospora crassa OR74A
61
62


Magnaporthe grisea 70-15
63
64


Gibberella zeae PH-1
65
66


Aspergillus niger
67
68


Neosartorya fischeri NRRL 181
69
70


(XP_001266525.1)




Neosartorya fischeri NRRL 181
71
72


(XP_001262996.1)




Aspergillus niger (An03g04520)
73
74


Aspergillus niger (An14g03280)
75
76


Aspergillus terreus NIH2624
77
78


Aspergillus clavatus NRRL 1
79
80


Aspergillus nidulans FGSC A4
81
82


Aspergillus oryzae
83
84


Ajellomyces capsulatus NAm1
85
86


Coccidioides immitis RS
87
88


Botryotinia fuckeliana B05.10
89
90


Phaeosphaeria nodorum SN15
91
92


Pichia guilliermondii ATCC 6260
95
96


Debaryomyces hansenii CBS767
97
98


Lodderomyces elongisporus NRRL
99
100


YB-4239




Vanderwaltozyma polyspora DSM
109
110


70294




Ashbya gossypii ATCC 10895
115
116


Laccaria bicolor S238N-H82
117
118


Coprinopsis cinerea okayama7#130
119
120


Cryptococcus neoformans var.
121
122


neoformans JEC21




Ustilago maydis 521
123
124


Malassezia globosa CBS 7966
125
126


Aspergillus clavatus NRRL 1
127
128


Neosartorya fischeri NRRL 181
129
130


(Putative)




Aspergillus oryzae
131
132


Aspergillus niger (An18g04160)
133
134


Aspergillus terreus NIH2624
135
136


Coccidioides immitis RS
137
138


(CIMG_04591)




Paracoccidioides brasiliensis
139
140


Phaeosphaeria nodorum SN15
141
142


Gibberella zeae PH-1
143
144


Neurospora crassa OR74A
145
146


Coprinopsis cinerea okayama 7#130
147
148


Laccaria bicolor S238N-H82
149
150


Ustilago maydis 521
151
152
















TABLE 3







Expression genes










SEQ ID NO:
SEQ ID NO:


Description
Nucleic acid
Peptide












Roseburia inulinivorans (RdhtA)
15
43


Roseburia inulinivorans (RdhtB)
16
44


Bacillus subtilis (alsS)
27
28


Vibrio cholerae (KARI)
35
36


Pseudomonas aeruginosa PAO1 (KARI)
37
38


Pseudomonas fluorescens PF5 (KARI)
39
40


Achromobacter xylosoxidans (sadB)
41
42


B12-independent glycerol dehydratase
190
191


from Clostridium butyricum




B-12 independent butanediol
192
193


dehydratase reactivase from




Clostridium butyricum









SEQ ID NO:17 is a synthetic rdhtAB sequence.
SEQ ID NOs:18-21 and 30-33 are primers for PCR, cloning or sequencing analysis used a described in the Examples herein.
SEQ ID NO:22 is a dual terminator sequence.
SEQ ID NO:23 is the Saccharomyces cerevisiae ADH terminator.
SEQ ID NO:24 is the Saccharomyces cerevisiae CYC1 terminator.
SEQ ID NO:25 is the Saccharomyces cerevisiae FBA promoter.
SEQ ID NO:26 is the Saccharomyces cerevisiae GPM promoter.
SEQ ID NO:29 is the pNY13 vector.
SEQ ID NO:34 is the Saccharomyces cerevisiae CUP1 promoter.
SEQ ID NO:173 is the codon optimized coding region for ILV3 DHAD from Kluyveromyces lactis.







TABLE 4







Functionally verified DHADs used for Profile HMM










SEQ ID NO:
SEQ ID NO:


Organism
Nucleic acid
Peptide












Nitrosomonas europaea ATCC 19718
174
175


Synechocystis sp. PCC 6803
176
177


Streptococcus mutans UA159
178
179


Streptococcus thermophilus LMG
180
181


18311




Ralstonia metallidurans CH34
182
183


Ralstonia eutropha JMP134
184
185


Lactococcus lactis subsp. cremoris
186
187


SK11




Flavobacterium johnsoniae UW101
188
189









DETAILED DESCRIPTION OF THE INVENTION
Disclosed herein is the discovery that introduced Fe—S containing proteins in yeast host cells have high activity levels when expression of endogenous Fe—S containing proteins is inhibited or disrupted. The present invention relates to recombinant yeast cells engineered to provide expression of at least one heterologous protein that is an Fe—S cluster protein, and engineered for reduced expression of at least one endogenous Fe—S cluster protein. In these cells the activity of the heterologous Fe—S cluster protein is improved, such that there is improved production of a product made in a biosynthetic pathway that includes the enzyme activity. Examples of commercially useful products from a pathway including an Fe—S protein include valine, isoleucine, leucine, pantothenic acid, iosbutanol, 2-butanone and 2-butanol.
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 “Fe—S cluster protein” is a protein that binds an iron-sulfur cluster and requires the binding of the cluster for its activity.
The term “2Fe-2S DHAD” refers to DHAD enzymes requiring a bound [2Fe-2S]2+ cluster for activity.
The term “Fe—S propanediol dehydratase reactivase” refers to propanediol dehydratase reactivases requiring a bound Fe—S cluster for activity.
The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.
The term “2-butanol biosynthetic pathway” refers to an enzyme pathway to produce 2-butanol from pyruvate.
The term “2-butanone biosynthetic pathway” refers to an enzyme pathway to produce 2-butanone from pyruvate.
There term “Dihydroxy-acid dehydratase”, also abbreviated DHAD, will refer to an enzyme that converts 2,3-dihydroxyisovalerate to α-ketoisovalerate.
The term “butanediol dehydratase”, also known as “diol dehydratase” or “propanediol dehydratase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone. Butanediol dehydratases that do not utilize the cofactor adenosyl cobalamin (also known as coenzyme B12, or vitamin B12; although vitamin B12 may refer also to other forms of cobalamin that are not coenzyme B12) are coenzyme B12-independent diol dehydratases that require association with a diol dehydratase reactivase that is a Fe—S cluster protein. Examples of B12-independent diol dehydratases include those from Clostridium glycolicum (Hartmanis et al. (1986) Arch. Biochem. Biophys. 245:144-152), Clostridium butyricum (protein SEQ ID NO:191; coding region SEQ ID NO:190; O'Brien et al. (2004) Biochemistry 43:4635-4645), and Roseburia inulinivorans (coding: SEQ ID NO:15; protein: SEQ ID NO:43; disclosed in co-pending US Patent Pub No. US20090155870.
The term “propanediol dehydratase reactivase”, also known as “diol dehydratase reactivase” or “butanediol dehydratase reactivase” refers to a reactivating factor for diol dehydratase, an enzyme which undergoes suicide inactivation during catalysis. Diol dehydratase reactivases associated with coenzyme B12-independent diol dehydratases may be Fe—S cluster proteins. Examples include those from Clostridium glycolicum (Hartmanis et al. (1986) Arch. Biochem. Biophys. 245:144-152), Clostridium butyricum (protein SEQ ID NO:193; coding region SEQ ID NO:192; O'Brien et al. (2004) Biochemistry 43:4635-4645), and Roseburia inulinivorans (coding: SEQ ID NO:16; protein: SEQ ID NO:44; disclosed in commonly owned and co-pending US Patent Pub No. US20090155870).
The term “reduced expression” as it applies to the expression of a protein in a cell host will include those situations where the activity of the protein is diminished as compared with a wildtype form (as with antisense technology for example) or substantially eliminated as with gene disruption, deletion or inactivation for example.
The term “carbon substrate” or “fermentable carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.
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 it 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 a foreign gene 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.
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 effecting 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 an 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 BLASTTM (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 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: NJ (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: 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, BLAST X (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR software (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher software (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.).
Discovery of Improved Fe—S Cluster Protein Activity in Yeast
Proteins that contain a bound iron-sulfur cluster (Fe—S) that is required for their activity may have low activity when used in heterologous expression systems. Formation of Fe—S clusters and their transfer to apo-proteins is a multistep process involving at least several proteins including cysteine desulfurase, a scaffold protein and a chaperone. Thus a heterologous Fe—S protein may not be effectively composed by the endogenous host system. Applicants have discovered a way to increase activity of an Fe—S protein expressed as a heterologous protein in a yeast host cell. Applicants have found that by reducing production of an endogenous Fe—S protein in the yeast host cell, an improvement in activity of an expressed heterologous Fe—S cluster protein can be achieved. Expression in yeast of either heterologous fungal or plant 2Fe-2S dihydroxy-acid dehydratase (DHAD) or Fe—S propanediol dehydratase reactivase (RdhtB) was improved when an endogenous gene encoding isopropylmalate dehydratase (LEU1) or an endogenous gene encoding dihydroxy-acid dehydratase (ILV3) was inactivated in the yeast host cells.
In yeast host cells with inactivation of a gene encoding an endogenous Fe—S protein, the activity of the expressed heterologous Fe—S protein may be increased to at least about 1.4 fold of the activity in a yeast host cell with no inactivation of Fe—S protein encoding gene. For example, the Kluyveromyces lactis DHAD had 1.4 fold activity in a LEU1 deletion host as compared to a host without the deletion; the Roseburia inulinivorans RdhtB had 1.7 fold comparative activity in a LEU deletion host as measured by the activated RdhtA protein activity (described below); Saccharomyces cerevisiae DHAD expressed in the cytosol had 1.5 fold comparative activity in a mitochondrial ILV3 deletion host; and Kluyveromyces lactis DHAD expressed in the cytosol had 7.4 fold comparative activity in a mitochondrial ILV3 deletion host.
Yeast Host, Cells with Reduced Expression of Endogenous Fe—S Protein
Reduced endogenous Fe—S protein expression may be engineered in any yeast cell that is amenable to genetic manipulation. Examples include yeasts of 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. Particularly suitable is Saccharomyces cerevisiae. 
In any of these yeasts, any endogenous Fe—S protein may be a target for reduced expression. Fe—S proteins in yeast that may be targeted for reduced expression include, for example, the following proteins (with encoding gene): aconitase (ACO1), homoaconitase (LYS4), DHAD (ILV3), lipoate synthase (LIP5), biotin synthase (BIO2), ferredoxin maturation (YAH1), NADH ubiquinone oxidoreductase (NDI1), succinate dehydrogenase (SDH2), ubiquinol-cytochrome-c reductase (RIP1), isopropylmalate isomerase (LEU1), sulfite reductase (ECM17), glutamate dehydrogenase (GLT1), ABC protein Rli1 (RLI1), NTPase Nbp35 (NBP35), and hydrogenase-like protein (NARI1). Yeast cells with reduced expression of individual Fe—S proteins may require special conditions for growth such as supplementation of the growth medium with a particular nutrient, as is well known to one skilled in the art. For example, a strain with disruption of LEU1 is supplemented with leucine, a strain with disruption of DHAD is supplemented with leucine, isoleucine, and valine, and a strain with disruption of LYS4 is supplemented with lysine. Some strains with a disruption require no supplementation for growth. Particularly suitable Fe—S proteins that may be targeted for reduced expression include Isopropylmalate isomerase (LEU1), Dihydroxyacid dehydratase (ILV3), Sulfite reductase (ECM17), Glutamate dehydrogenase (GLT1), and Biotin synthase (BIO2). Reduced expression is engineered for at least one endogenous Fe—S protein, and two or more endogenous Fe—S proteins may be reduced.
LEU1 encodes isopropylmalate dehydratase, an enzyme belonging to EC 4.2.1.33 that is involved in branched chain amino acid biosynthesis, specifically synthesis of leucine. Any gene encoding an isopropylmalate dehydratase, which is an enzyme requiring a 4Fe-4S cluster for activity, may be inactivated in a yeast host cell of this disclosure. Examples of yeast LEU1 inactivation target genes and their encoded proteins are those from Saccharomyces cerevisiae (coding SEQ ID NO:1; protein SEQ ID NO:2), Schizosaccharomyces pombe (coding SEQ ID NO:3; protein SEQ ID NO:4), Candida galbrata strain CBS138 (coding SEQ ID NO:5; protein SEQ ID NO:6), Candida albicans SC5314 (coding SEQ ID NO:7; protein SEQ ID NO:8), Kluyveromyces lactis (coding SEQ ID NO: protein SEQ ID NO:10), Yarrowia lipolytica (coding SEQ ID NO:11; protein SEQ ID NO:12) and Pichia stipitis (coding SEQ ID NO:13: protein SEQ ID NO:14).
Similarly in any of the yeast hosts described herein, an endogenous ILV3 gene may be inactivated to reduce endogenous Fe—S protein expression. ILV3 encodes mitochondrial DHAD that is involved in branched chain amino acid biosynthesis. Mitochondrial DHAD is encoded by a nuclear gene, and has a mitochondrial targeting signal sequence so that it is transported to and localized in the mitochondrion. Any ILV3 gene may be inactivated in a yeast host cell of this disclosure. Examples of yeast ILV3 inactivation target genes and their encoded proteins are those from Saccharomyces cerevisiae YJM78 (coding SEQ ID NO:111; protein SEQ ID NO:112), Schizosaccharomyces pombe (coding SEQ ID NO:93; protein SEQ ID NO:94), Candida galbrata strain CBS138 (coding SEQ ID NO:107; protein SEQ ID NO:108), Candida albicans SC5314 (coding SEQ ID NO:101; protein SEQ ID NO:102), Kluyveromyces lactis (coding SEQ ID NO:113; protein SEQ ID NO:114), Yarrowia lipolytica (coding SEQ ID NO:105; protein SEQ ID NO:106) and Pichia stipitis CBS 6054 (coding SEQ ID NO:103; protein SEQ ID NO:104).
Because genes encoding isopropylmalate dehydratases and DHAD enzymes genes are well known, and because of the prevalence of genomic sequencing, additional suitable species of these enzymes can be readily identified by one skilled in the art on the basis of sequence similarity using bioinformatics approaches. Typically BLASTTM (described above) searching of publicly available databases with known isopropylmalate dehydratase amino acid sequences, such as those provided herein, is used to identify these enzymes and their encoding sequences that may be targeted for inactivation in the present strains. For example, endogenous yeast isopropylmalate dehydratase and DHAD proteins having amino acid sequence identities of at least about 70-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or 98% sequence identity to any of the isopropylmalate dehydratase proteins of SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14 and the DHAD proteins of SEQ ID NOs:94, 102, 104, 106, 108, 112, and 114 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.
Additionally, the sequences of LEU1 coding regions and ILV3 provided herein may be used to identify other homologs in nature. For example each of the coding regions 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 isopropylmalate dehydratase and DHAD 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.).
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 provided isopropylmalate dehydratase and DHAD encoding sequences can 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 polymer (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.
Protein and nucleic acid encoding sequences for any of the other Fe—S proteins that may be targeted for reduced activity in a yeast cell of the invention may be identified using bioinformatics and other methods well known to one skilled in the art. For example, aconitase sequences are identified by keyword searching in bioinformatics databases. Several sequences identified by this method are those from Saccharomyces cerevisiae (coding SEQ ID NO:153; protein SEQ ID NO:154), Schizosaccharomyces pombe on chromosome II (coding SEQ ID NO:155; protein SEQ ID NO:156), Schizosaccharomyces pombe on chromosome I (coding SEQ ID NO:157; protein SEQ ID NO:158), Kluyveromyces lactis (coding SEQ ID NO:15; protein SEQ ID NO:160), Candida albicans SC5314 (coding SEQ ID NO:161; protein SEQ ID NO:162), Yarrowia lipolytica (coding SEQ ID NO:163; protein SEQ ID NO:164), Pichia stipitis CBS 6054 (coding SEQ ID NO:165; protein SEQ ID NO:166), Candida galbrata CBS138 chromosome F (coding SEQ ID NO:167; protein SEQ ID NO:168), Candida galbrata CBS138 chromosome D (coding SEQ ID NO:169; protein SEQ ID NO:170), and Candida galbrata CBS138 chromosome K (coding SEQ ID NO:171; protein SEQ ID NO:172).
Genes encoding Fe—S proteins, for example LEU1, ILV3, or ACO1 may be disrupted in any yeast cell using genetic modification. 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. All of these methods may be readily practiced by one skilled in the art making use of the known or identified coding sequences such as LEU1 or ILV3.
DNA sequences surrounding a LEU1, ILV3, or ACO1 coding sequence are also useful in some modification procedures and are available for yeasts such as for Saccharomyces 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 GRID 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, such as LEU1 or ILV3, 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 encided activity. Using this type of method, the DNA sequence of for example the LEU1, ILV3, or any other region of the genome affecting expression of a target Fe—S 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 Fe—S protein activity.
Heterologous Fe—S Proteins
Any fungal or plant 2Fe-2S cluster dihydroxy-acid dehydratase (DHAD) and any Fe—S propanediol dehydratase reactivase may be expressed as a heterologous protein in a yeast host cell engineered as disclosed herein for reduced endogenous Fe—S cluster protein expression, and increased activity may be obtained. A heterologous protein includes one that is expressed in a manner differently from expression of a corresponding endogenous protein. For example in yeast, endogenous DHAD is encoded by ILV3 in the nucleus and the expressed DHAD protein has a mitochondrial targeting signal sequence such that the protein is localized in the mitochondrion. An Fe—S cluster is added to the DHAD protein in the mitochondrion for its activity in branched chain amino acid biosynthesis. It is desirable to express DHAD activity in the cytosol for participation in biosynthetic pathways that are localized in the cytosol. Cytosolic expression of DHAD in yeast is heterologous expression since the native protein is localized in the mitochondrion. For example, heterologous expression of the Saccharomyces cerevisiae DHAD in S. cerevisiae is obtained by expressing the S. cerevisiae DHAD coding region with the mitochondrial targeting signal removed, such that the protein remains in the cytosol. 2Fe-2S DHADs that may be used in the present disclosure include those from fungi and plants. Representative fungal or plant 2Fe-2S DHADs are listed in Tables 1 and 2. Fungal or plant 2Fe-2S DHADs with amino acid sequence identities of 95% or greater were removed from the analysis providing this list for simplification. However, any sequences with 95% or greater amino acid identities to any of these sequences are useful in the present invention. The analysis used to obtain 2Fe-2S DHADs is described in commonly owned and co-pending U.S. Patent Application 61/100,792, which is herein incorporated by reference. The analysis is as follows: 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 SEC) ID NO:174; Protein SEQ ID NO:175), Synechocystis sp. PCC6803 (DNA SEQ ID:176; Protein SEQ ID NO:177), Streptococcus mutans (DNA SEQ ID NO:178; Protein SEQ ID NO:179), Streptococcus thermophilus (DNA SEQ ID NO:180; protein SEQ ID No:181), Ralstonia metallidurans (DNA SEQ ID NO:182; protein SEQ ID NO:183), Ralstonia eutropha (DNA SEQ ID NO:184; protein SEQ ID NO:185), and Lactococcus lactis (DNA SEQ ID NO:186; protein SEQ ID NO:187). In addition the DHAD from Flavobacterium johnsoniae (DNA SEQ ID NO:188; protein SEQ ID NO:189) 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, given in Table 9.
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, arabonate 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]2+ cluster. Proteins having the three conserved cysteines include arabonate dehydratases and 2Fe-2S DHADs. The 2Fe-2S DHADs may be distinguished from the arabonate dehydratases by analyzing for signature conserved amino acids found to be present in the 2Fe-2S DHADs or in the arabonate 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 arabonate dehydratases, respectively, at the following positions (with greater than 90% occurrence): 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.
The proteins identified by this process that have a fungal or plant origin, such as SEQ ID NOs:46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150 and 152 may be used in the present invention, as well as any protein with amino acid identity of at least about 95%, 96%, 97%, 98%, or 99% to any of these sequences. Particularly suitable is the DHAD from Kluyveromyces lactis (SEQ ID NO:114) and DHADs with at least about 90% amino acid sequence identity to SEQ ID NO:114 using 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.
In addition, fungal or plant 2Fe-2S DHADs that may be used in the present invention may be identified by their position in a fungal or plant 2Fe-2S DHAD branch of a phylogenetic tree of DHAD related proteins. In addition, 2Fe-2S DHADs that may be used may be identified using sequence comparisons with any of the fungal or plant 2Fe-2S DHADs whose sequences are provided herein, where sequence identity may be at least about 80%-85%, 85%-90%, 90%-95% or 95%-99%.
Additionally, the sequences of fungal or plant 2Fe-2S DHADs provided herein may be used to identify other homologs in nature. For example each of the DHAD encoding nucleic acid fragments given herein as SEQ ID NOs:45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 11, 121, 123, 125, 127, 129, 131, 133, 135, 137, 13, 141, 143, 145, 147, 149 and 151 may be used to isolate genes encoding homologous proteins as described above for the LEU1 coding region.
The coenzyme B12-independent propanediol deydratase reactivase of Roseburia inulinivorans is a protein requiring an Fe—S cluster for activity. This protein, RdhtB, is disclosed in co-pending US Patent Pub No. US20090155870, which is herein incorporated by reference. RdhtB reactivates a coenzyme B12-independent propanediol deydratase of Roseburia inulinivorans, which is named RdhtA and is also disclosed in commonly owned and co-pending US Patent Pub No. US20090155870. The activity of RdhtB may be assessed by assaying the activity of RdhtA, since RdhtB is required for RdhtA activity. Activity of RdhtB, and therefore of RdhtA, is improved by expressing in a yeast host with reduced endogenous Fe—S protein expression disclosed herein. Heterologous expression of any coenzyme B12-independent propanediol deydratase reactivase that requires an Fe—S cluster for activity may be improved in a yeast strain having reduced endogenous Fe—S protein expression. A coenzyme B12-independent propanediol deydratase reactivase may be readily identified by one skilled in the art by assessing propanediol deydratase activity of the associated propanediol deydratase enzyme in the presence or absence of coenzyme B12. An example is a diol dehydratase reactivase of Clostridium butyricum (coding region SEQ ID NO:192; protein SEQ ID NO:193).
Other coenzyme B12-independent propanediol deydratase reactivases that may be used may be identified through bioinformatics analysis of sequences as compared to that of RdhtB SEQ ID NO:44 by one skilled in the art. Proteins with coenzyme B12-independent propanediol deydratase reactivase activity and sequence identity to SEQ ID NO:44 of at least about 80%-85%, 85%-90%, 90%-95% or 95%-99% may be used. Particularly suitable are those that are at least about 90% identical to the amino acid sequence as set forth in SEQ ID NO:44 using 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.
In addition, other coenzyme B12-independent propanediol deydratase reactivase homologs that may be used may be identified using the RdhtB coding region (SEQ ID NO:16) by methods as described above for the LEU1 coding region.
Expression of Heterologous Fe—S Proteins
Expression is achieved by transforming with a sequence encoding an Fe—S protein. The coding region to be expressed may be codon optimized for the target host cell, as well known to one skilled in the art. Methods for gene expression in yeast 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 in yeast, including, but not limited to promoters derived from the following genes: CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1, FBA, GPD, GPM, and AOX1. Suitable transcriptional terminators include, but are not limited to FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1, and ADH1.
Suitable promoters, transcriptional terminators, and coding regions may be cloned into E. coli-yeast shuttle vectors, and transformed into yeast cells. These vectors allow strain propagation in both E. coli and yeast strains. Typically the vector used 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, Manassas, Va.), 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 the described Fe—S protein coding regions 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.
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.
Product Biosynthesis with Improved Heterologous Fe—S Protein Activity
Production of any product that has an Fe—S protein contributing to its biosynthetic pathway may benefit from the improved activity disclosed herein of a heterologous expressed Fe—S protein in a yeast host with reduced endogenous Fe—S protein expression. For example, DHAD provides a step in pathways for biosynthesis of isobutanol, and RdhtB contributes to a biosynthetic pathway to produce 2-butanone or 2-butanol.
Biosynthetic pathways including a step performed by DHAD for synthesis of isobutanol are disclosed in commonly owned and co-pending US Patent Application publication US 20070092957 A1, which is herein incorporated by reference. A diagram of the disclosed isobutanol biosynthetic pathways is provided in FIG. 1. Production of isobutanol in a strain disclosed herein benefits from increased DHAD activity. As described in US Patent Pub No. US20070092957 A1, steps in an example isobutanol biosynthetic pathway include conversion of:

    
    
        pyruvate to acetolactate (FIG. 1 pathway step a) as catalyzed for example by acetolactate synthase;
        acetolactate to 2,3-dihydroxyisovalerate (FIG. 1 pathway step b) as catalyzed for example by acetohydroxy acid isomeroreductase;
        2,3-dihydroxyisovalerate to α-ketoisovalerate (FIG. 1 pathway step c) as catalyzed for example by acetohydroxy acid dehydratase also called DHAD;
        α-ketoisovalerate to isobutyraldehyde (FIG. 1 pathway step d) as catalyzed for example by branched-chain α-keto acid decarboxylase; and
        isobutyraldehyde to isobutanol (FIG. 1 pathway step e) as catalyzed for example by branched-chain alcohol dehydrogenase.
    
    


The substrate to product conversions, and enzymes involved in these reactions, for steps f, g, h, I, j, and k of alternative pathways are described in US 20070092957 A1.
Genes that may be used for expression of the enzymes for the isobutanol pathways are described in US 20070092957 A1, and additional genes that may be used can be identified by one skilled in the art through bioinformatics or experimentally as described above. The preferred use in all three pathways of ketol-acid reductoisomerase (KARI) enzymes with particularly high activities are disclosed in commonly owned and co-pending US Patent Pub No. US20080261230. Examples of high activity KARIs disclosed therein are those from Vibrio cholerae (DNA: SEQ ID NO:35; protein SEQ ID NO:36), Pseudomonas aeruginosa PA01, (DNA: SEQ ID NO:37; protein SEQ ID NO:38), and Pseudomonas fluorescens PF5 (DNA: SEQ ID NO:39; protein SEQ ID NO:40).
Additionally described in US 20070092957 A1 are construction of chimeric genes and genetic engineering of yeast, exemplified by Saccharomyces cerevisiae, for isobutanol production using the disclosed biosynthetic pathways.
A biosynthetic pathway including propanediol dehydratase for synthesis of 2-butanone and 2-butanol is disclosed in commonly owned and co-pending US Patent Pub No. US20070292927A1, which is herein incorporated by reference. A diagram of the disclosed 2-butanone and 2-butanol biosynthetic pathway is provided in FIG. 2. 2-Butanone is the product made when the last depicted step of converting 2-butanone to 2-butanol is omitted. Production of 2-butanone or 2-butanol in a strain disclosed herein benefits from increased coenzyme 812-independent propanediol dehydratase reactivase activity. As described in US Patent Pub No. US20070292927A1, steps in the disclosed biosynthetic pathway include conversion of:

    
    
        pyruvate to acetolactate (FIG. 2 step a) as catalyzed for example by acetolactate synthase;
        acetolactate to acetoin (FIG. 2 step b) as catalyzed for example by acetolactate decarboxylase;
        acetoin to 2,3-butanediol (FIG. 2 step i) as catalyzed for example by butanediol dehydrogenase;
        2,3-butanediol to 2-butanone (FIG. 2 step j) as catalyzed for example by diol dehydratase glycerol dehydratase, or propanediol dehydratase; and
        2-butanone to 2-butanol (FIG. 2 step f) as catalyzed for example by butanol dehydrogenase.
    
    


Genes that may be used for expression of these enzymes are described in US Patent Pub No. US20070292927A1. The use in this pathway in yeast of the butanediol dehydratase from Roseburia inulinivorans, RdhtA, (protein SEQ ID NO:43, coding region SEQ ID NO:15) is disclosed in commonly owed and co-pending US Patent Pub No. US20090155870. This enzyme is used in conjunction with the butanediol dehydratase reactivase from Roseburia inulinivorans, RdhtB, (protein SEQ ID NO:44, coding region SEQ ID NO:16). This butanediol dehydratase is desired in many hosts because it does not require coenzyme B12.
Additionally described in US Patent Pub No. US20090155870 are construction of chimeric genes and genetic engineering of yeast for 2-butanol production using the US 20070292927A1 disclosed biosynthetic pathway.
Fermentation Media
Yeasts disclosed herein may be grown in fermentation media for production of a product having an Fe—S protein as part of the biosynthetic pathway. Fermentation media must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose 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. 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 yeast 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 and will only be limited by the choice of organism.
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.
In addition to an appropriate carbon source, fermentation media must contain 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.
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 butanol 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.
The present invention may be practiced using either batch, fed-batch or continuous processes and known modes of fermentation are suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for 1-butanol production.
Methods for Butanol Isolation from the Fermentation Medium
Bioproduced butanol 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 butanol 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 butanol 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 butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol 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 butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the butanol. In this method, the butanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the butanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The butanol-rich decanted organic phase may be further purified by distillation in a second distillation column.
The butanol may also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the butanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The butanol-containing organic phase is then distilled to separate the butanol from the solvent.
Distillation in combination with adsorption may also be used to isolate butanol from the fermentation medium. In this method, the fermentation broth containing the butanol 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 butanol from the fermentation medium. In this method, the fermentation broth containing the butanol 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. 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.
HPLC
Analysis for fermentation by-product composition is well known to those skilled in the art. For example, one high performance liquid chromatography (HPLC) method utilizes a SHODEX SH-1011 column with a SHODEX SH-G guard column (both available from Waters Corporation, Milford, Mass.), with refractive index (RI) detection. Chromatographic separation is achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanol retention time is 47.6 minutes.
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, and “GC” means gas chromatography. The term “molar selectivity” is the number of moles of product produced per mole of sugar substrate consumed and is reported as a percent.
Example 1
Expression of DHAD from K. lactis in LEU1 Deletion Strain of S. cerevisiae 
The yeast LEU1 gene encodes isopropylmalate dehydratase, an enzyme that requires an Fe—S cluster for its function. The impact of LEU1 deletion on DHAD activity expressed from the Kluyveromyces lactis DHAD coding region was examined in this example. For gene expression in yeast, the shuttle vector pNY13 (SEQ ID NO:29) derived from pRS423 was used. This 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. pNY9 is the same vector with a URA3 marker replacing the HIS3 marker.
The ILV3 coding region for DHAD from Kluyveromyces lactis was synthesized with codon-optimization for expression in S. cerevisiae by DNA 2.0 (Menlo Park, Calif.). The cloned synthesized sequence was PCR amplified. During amplification, a portion of the mitochondrial signal peptide for the DHAD at the N-terminus was deleted by using ilv3(K)(0)-F(delet) as the forward primer with ilv3(K)(o)-R as the reverse primer, resulting in a coding region for cytoplasmic expression (SEQ ID NO:173). In addition, an SphI site was incorporated in the forward primer, while a NotI site was included in the reverse primer. The PCR product was cloned into the shuttle vectors pNY9 and pNY13 so that the ILV3 coding region was under the control of the FBA promoter. Both PCR product and each vector (pNY9, pNY13) were digested with SphI and NotI. After digestion, the components were ligated, and the ligation mixture was transformed into TOP10 competent cells (Invitrogen). Transformants were selected in LB agar plates supplemented with 100 μg/ml of ampicillin. Positive clones were screened by PCR with the forward and reverse primers described above. The resulting plasmids were designated as pRS423::FBAp-ILV3(KL) and pRS426::FBAp-ILV3(KL), derived from plasmids pNY13 and pNY9, respectively.
To study the expression of the DHAD from K. lactis in S. cerevisiae the expression vector pRS423::FBAp-ILV3(KL) along with an empty vector pRS426 were transformed into strains BY4743 and BY4743 leu1::kanMX4 (ATCC 4034377). The competent cell preparation and transformation were based on the Frozen Yeast Transformation kit from Zymo Research. The transformants were selected on agar plates with yeast synthetic medium lacking histidine and uracil (Teknova). For enzymatic assays, the strains carrying the expression construct and the empty vector pRS426 were first grown overnight in 5 ml synthetic complete yeast medium lacking histidine and uracil. The 5 ml overnight cultures were transferred into 100 ml of medium in a 250 ml flask. The cultures were harvested when they reached 1 to 2 O.D. at 600 nm. The samples were washed with 10 ml of 20 mM Tris (pH 7.5) and then resuspended in 1 ml of the same Tris buffer. The samples were transferred into 2.0 ml tubes containing 0.1 mm silica (Lysing Matrix B, MP biomedicals). The cells were then broken in a bead-beater (BIO101). The supernatant was obtained by centrifugation in a microfuge at 13,000 rpm at 4° C. for 30 minutes. Typically, 0.06 to 0.1 mg of crude extract protein was used in a DHAD assay. Protein in the crude extracts was determined by Bradford assay with Coomassie stain.
Dihydroxy-Acid Dehydratase Enzyme Assay
The in vitro DHAD enzyme assay is a variation on the assay described in Flint et al. (J. Biol. Chem. (1993) 268:14732-14742.). The assay was performed in a 1.6 ml total volume and consisted of: 800 μl 2× buffer (100 mM Tris pH 8.0, 20 mM MgCl2), 160 μl 10× substrate (15.6 mg/ml dihydroxyisovalerate), crude extract (typically 50-200 μg protein), and water. The reaction was incubated at 37° C. At 0, 30, 60, and 90 minute time intervals, 350 μl aliquots of the reaction were removed and incubated with 350 μl of 0.05% dinitrophenyhydrazine in 1N HCl for 30 minutes at 25° C. To quench the reaction, 350 μl of 4N sodium hydroxide was added to the reaction mixture, and the reaction was centrifuged at 15,000×g for 2 minutes. The supernatant was transferred to a plastic disposable cuvette and absorbance at 540 nm was measured in a spectrophotometer. The amount of α-ketoisovalerate (HIV) produced was determined by entering the absorbance into the linear regression equation obtained from a standard curve of α-ketoisovalerate. The amount of KIV produced at each time point was plotted to determine the rate of production. The slope of the linear regression was then used to calculate specific activity using the formula:

Specific activity calculation=(slope of KIV production/1000)/mg protein per 1.6 mL reaction=mmol/min*mg

The dehydratase from K. lactis had a specific activity in the range of 0.2 to 0.35 μmol min−1 mg−1 when expressed in yeast strain BY4743 (Δleu1). In contrast, this enzyme had a specific activity in the range of only 0.14 μmol min−1 mg−1 when expressed in the parent yeast strain BY4743. Strains BY4743 (Δleu1) and wildtype BY4743 containing empty vectors pRS423 or pRS426 had a background of activity in the range of 0.03 to 0.1 μmol min−1 mg−1.
Example 2
Expression of Diol Dehydratase in LEU1 Deletion Strain of S. cerevisiae 
A coenzyme B12-independent propanediol dehydratase is disclosed in commonly owned and co-pending US Patent Pub No. US20090155870. The sequences encoding this coenzyme B12-independent (S-adenosylmethionine (SAM)-dependent) propanediol dehydratase (SEQ ID NO:15) and its putative associated reactivase (SEQ ID NO:16) in the bacterium Roseburia inulinivorans [Scott et al. (2006) J. Bacteriol. 188:4340-9], hereafter referred to as rdhtA and rdhtB, respectively, were synthesized as one DNA fragment (SEQ ID NO:17) by standard methods and cloned into an E. coli vector (by DNA2.0, Inc., Menlo Park, Calif.). This clone was used as a PCR template to prepare separate RdhtA and RdhtB coding region fragments. The RdhtA coding region for the diol dehydratase was amplified by PCR using primers N695 and N696 (SEQ ID NOs:18 and 19). The RdhtB coding region for the diol dehydratase activase, was amplified by PCR using primers N697 and N698 (SEQ ID NOs:20 and 21). The two DNA fragments were combined with a dual terminator DNA fragment (SEQ ID NO:22) that has an ADH terminator (SEQ ID NO:23) and a CYC1 terminator (SEQ ID NO:24) adjacent to each other in opposing orientation using SOE PCR (Horton et al. (1989) Gene 77:61-68). The dual terminator fragment was isolated as a 0.6 kb fragment following PacI digestion of pRS426::FBA-ILV5+GPM-kivD (described in co-owned and co-pending US Patent Publication 20070092957 A1, Example 17). The resulting 4 kb DNA fragment had the RdhtA and RdhtB coding regions in opposing orientation on either side of the dual terminator, with the 3′ end of each coding region adjacent to the dual terminator sequence. This DNA fragment was then cloned by gap repair methodology (Ma et al. (1987) Genetics 58:201-216) into the S. cerevisiae shuttle vector pRS426::FBA-ILV5+GPM-kivD that was prepared by digestion with BbvCI to remove the ILV5 and kivD coding regions and dual terminator sequence between their 3′ ends. The resulting plasmid, pRS426::RdhtAB (below), contained the RdhtA gene under the control of the S. cerevisiae FBA promoter (SEQ ID NO:25) and the RdhtB gene under control of the S. cerevisiae GPM promoter (SEQ ID NO:26).
Plasmids pRS426 and pRS426::RdhtAB were introduced into S. cerevisiae strains BY4743 (ATCC 201390) and BY4743 leu1:kanMX4 (ATCC 4034377) by standard techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Cells were plated on synthetic complete medium lacking uracil to select for transformants. Transformants were tested for diol dehydratase activity using an in vivo assay as follows. Patched cells grown on solid medium were used to inoculate liquid media (20 ml) in petri plates. Media used were synthetic complete minus uracil with and without addition of 5 g/L 1,2-propanediol 9Aldrich Cat. No. 398039). The petri plates were transferred to an AnaeropackTM System jar (Mitsubishi Gas Chemical Co. Cat. No. 50-70). An anaerobic environment (<0.1% oxygen) was generated using PACK-ANAERO sachets (Mitsubishi Gas Chemical Co. Cat. No. 10-01). After 48 hours, culture supernatants were sampled, filtered and analyzed by HPLC as described in General Methods. Propanol, which has a retention time of 38.8 minutes, was observed in culture supernatants of strains carrying pRS426::RdhtAB when 1,2-propanediol was provided in the medium. The results given in Table 5 show that more propanol was produced in the supernatants of the strain also carrying the LEU1 deletion than in the strain without the LEU1 deletion. Statistical analysis gave a P score of less than 0.0005.







TABLE 5







Propanol production with propanediol dehydratase/reactivase


expression in yeast with and without LEU1 knockout.










1,2-propanediol
Propanol Peak


Strain
Added
Area





BY4743
5 g/L
19472 ± 1403


Δleu1::kanMX4/pRS426::RdhtAB

(n = 6)


BY4743
0 g/L
2478


Δleu1::kanMX4/pRS426::RdhtAB

(n = 1)


BY4743/pRS426::RdhtAB
5 g/L
11830 ± 1963




(n = 6)


BY4743/pRS426::RdhtAB
0 g/L
2369




(n = 1)


BY4743 Δleu1::kanMX4/pRS426
5 g/L
2633




(n = 1)


BY4743/pRS426
5 g/L
2841




(n = 1)









Example 3
Improving Cytosolic Dihydroxy-Acid Dehydratase (DHAD) Activity in S. cerevisiae Through a Disruption of Mitochondrial ILV3
Vector/Host Construction
In S. cerevisiae ILV3 encodes the mitochondrial dihydroxy-acid dehydratase that is involved in branched chain amino acid biosynthesis. To reduce background from endogenous ILV3 expression for in vitro enzymatic assays in S. cerevisiae, an ilv3::URA3 disruption cassette was constructed by PCR amplification of the URA3 marker from pRS426 (ATCC No. 77107) with primers “ILV3::URA3 F” and “ILV3::URA3 R”, given as SEQ ID NO:30 and 31. These primers produced a 1.4 kb URA3 PCR product that contained 70 bp 5′ and 3′ extensions identical to sequences upstream and downstream of the ILV3 chromosomal locus for homologous recombination. The PCR product was transformed into BY4741 cells (ATCC 201388) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and resulting transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR using primers “ILV3 F Check” and “URA3 REV Check”, given as SEQ ID NOs:32 and 33, to verify integration at the correct site and disruption of the endogenous ILV3 locus. The correct transformants had the genotype: BY4741 ilv3::URA3.
Construction of plasmid pRS423::FBAp-ILV3(KL) and pRS426::FBAp-ILV3(KL) were described in Example 1. Construction of pRS423::CUP1-alsS+FBA-ILV3 has been described in co-owned and co-pending US Patent Publication US20070092957 A1, Example 17 which is herein incorporated by reference. pRS423::CUP1-alsS+FBA-ILV3 is the same plasmid as pRS423::CUP1p-alsS-FBAp-ILV3. This construction contains a chimeric gene containing the S. cerevisiae CUP1 promoter (SEQ ID NO:34), alsS coding region from Bacillus subtilis (SEQ ID NO:27), and CYC1 terminator (SEQ ID NO:24); and also a chimeric gene containing the S. cerevisiae FBA promoter (SEQ ID NO:25), ILV3 coding region from S. cerevisiae lacking the mitochondrial targeting signal coding sequence (SEQ ID NO:111) and ADH1 terminator (SEQ ID NO:23).
Preparation of Samples
Plasmid vectors pRS423::CUP1p-alsS-FBAp-IL V3 and pRS423::FBAp-ILV3(KL) were transformed into strain BY4741 ilv3::URA3 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and maintained on synthetic complete media lacking histidine. Plasmid vectors pRS423::CUP1p-alsS-FBAp-ILV3 and pRS426::FBAp-ILV3(KL) were also transformed into strain BY4741. Aerobic cultures were grown in 1000 ml flasks containing 200 ml synthetic complete media lacking histidine and supplemented with 2% glucose in an INNOVA 4000 incubator (New Brunswick Scientific, Edison, N.J.) at 30° C. and 225 rpm. Cultures were harvested at OD600 measurements of 1.0-2.0 and pelleted by centrifugation at 6000×g for 10 minutes. Cell pellets were washed with 10 mM Tris-HCl, pH 8.0 and pellets were stored at −80° C. until assayed for activity. Cell free extracts were prepared by standard bead beating method using 1 ml of 0.5 mm beads and 1.5 ml of yeast cell suspension. Protein concentration in the extracts was determined by Bradford assay with Coomassie stain. DHAD enzyme assays and specific activity calculations were performed as described in Example 1. The results given in Table 6 show that there was higher DHAD activity in the ILV3 deletion cells than in cells without the ILV3 deletion.







TABLE 6







DHAD activity in yeast with and without ILV3 deletion.











Average



Specific
Specific



Activity
Activity


Strain
(μmol/min*mg)
(μmol/min*mg)












BY4741
0.018
0.013



0.014




0.008



BY4741 pRS423::CUP1p-alsS-FBAp-
0.018
0.019


ILV3





0.020



BY4741 pRS426::FBAp-ILV3(KL)
0.040
0.038



0.036



BY4741 ilv3::URA3
0.00006
0.00041



0.00075



BY4741 ilv3::URA3 pRS423::CUP1p-
0.030
0.029


alsS-FBAp-ILV3





0.028



BY4741 ilv3::URA3 pRS423::FBAp-
0.317
0.281


ILV3(KL)





0.244









Verification of Alpha-Ketoisovalerate Formation by HPLC

Formation of alpha-ketoisovalerate from the in vitro DHAD enzyme assays was accomplished using HPLC and semicarbizide derivatization. DHAD enzyme assays were performed in a 1.6 ml total volume and consisted of: 800 μl 2× buffer (100 mM Tris pH 8.0, 20 mM MgCl2), 160 μl 10× substrate (15.6 mg/ml dihydroxyisovalerate), crude extract (typically 50-200 μg protein), and water. The reactions were incubated at 37° C. At time intervals of zero and 90 minutes 350 μl aliquots of the reactions were removed, transferred to ice, and centrifuged at 13,000×g for 2 minutes at 4° C. to remove precipitated protein. The supernatants were transferred to ice-chilled MICROCON YM-10 (Sigma) spin columns and centrifuged at 13,000×g for 20 minutes at 4° C. to remove enzymes and soluble proteins. The flowthroughs were mixed with 100 μl derivatizing reagent (1% semicarbizide hydrochloride and 1.5% sodium acetate trihydrate) and incubated at room temperature for 15 minutes. The reactions were spun through COSTAR spin filters (CoStar, 0.22 μm filter) at 13,000×g for 5 minutes at 4° C. to remove any precipitates. The flowthroughs were transferred to HPLC vials for analysis.
Analysis of derivatized alpha-ketoisovalerate was conduced using reverse phase chromatography on a SUPELCOSIL LC-18 column with SUPELGUARD LC-18-DB guard column (Supelco; 25 cm×4.6 mm, 5 μm). Injection volumes were 10 μl. Mobile phases were methanol (A) and 50 mM NaOAc pH 7.2. The gradient program utilized is given in Table 7, with detection at 250 nm.







TABLE 7







Gradient used for derivatized alpha-ketoisovalerate HPLC assay











Time (min)
Flow (ml/min)
% NaOAc (50 mM)
% MEOH
Curve














Initial
1.0
95
5



 5
1.0
95
5
6


20
1.0
70
30
6


21
1.0
0
100
6


25
1.0
0
100
6


26
1.0
95
5
6


35
1.0
95
5
6





The retention time of semicarbizide-derivatized alpha-ketoisovalerate was 11.5 minutes.






The results, which are given in Table 8, confirmed that KIV was produced in the cells, as detected in the indirect assay for specific activity above. The amount of KIV listed in the DHAD Assay column is the amount determined indirectly in the 90 min sample for the activity assay described above in determining the specific activity. This amount of KIV correlates well with the amount detected in the HPLC assay.







TABLE 8







Comparison of KIV detected in DHAD activity assay and by HPLC.









keto-isovalerate production (μM)









Strain
DHAD Assay
HPLC





BY4741 ilv3::URA3 pRS423::FBAp-
256
256


ILV3(KL)




BY4741 ilv3::URA3 pRS423::FBAp-
162
163


ILV3(KL)

















TABLE 9







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.
































A
C
D
E
F
G
H
I
K











Position in


HMM
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
−3935
−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

## Claims

1. A method of converting 2,3-dihydroxyisovalerate to α-ketoisovalerate, comprising providing a recombinant yeast host cell expressing a heterologous dihydroxy-acid dehydratase (DHAD) protein that comprises
(i) an amino acid sequence having at least 95% identity to SEQ ID NO: 179 or 187; and
(ii) three conserved cysteine residues that correspond to positions 56, 129, and 201 of SEQ ID NO:179;

wherein the substrate 2,3-dihydroxyisovalerate is present in the yeast host cell and wherein the expressed heterologous DHAD protein catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate.

2. The method of claim 1, wherein the amino acid sequence of the heterologous DHAD protein comprises SEQ ID NO: 179.

3. The method of claim 1, wherein the amino acid sequence of the heterologous DHAD protein comprises SEQ ID NO: 187.

4. The method of claim 1, wherein the recombinant yeast host cell comprises a disruption in an ilv3 gene that encodes mitochondrial DHAD, resulting in reduced expression of endogenous mitochondrial DHAD.

5. The method of claim 1, wherein the recombinant yeast host cell comprises an engineered isobutanol biosynthetic pathway.

6. The method of claim 5, wherein the engineered isobutanol biosynthetic pathway comprises the following substrate to product conversions:
(i) pyruvate to acetolactate (pathway step a);
(ii) acetolactate of (i) to 2,3-dihydroxyisovalerate (pathway step b);
(iii) 2,3-dihydroxyisovalerate of (ii) to α-ketoisovalerate (pathway step c);
(iv) α-ketoisovalerate of (iii) to isobutyraldehyde (pathway step d); and
(v) isobutyraldehyde of (iv) to isobutanol (pathway step e),

and wherein
(a) the substrate to product conversion of step (i) is catalyzed by an acetolactate synthase;
(b) the substrate to product conversion of step (ii) is catalyzed by a ketol-acid reductoisomerase;
(c) the substrate to product conversion of step (iii) is catalyzed by the heterologous DHAD;
(d) the substrate to product conversion of step (iv) is catalyzed by an α-keto acid decarboxylase; and
(e) the substrate to product conversion of step (v) is catalyzed by an alcohol dehydrogenase.

7. The method of claim 6, wherein two or more of the enzymes (a), (b), and (d) are heterologous to the recombinant yeast host cell.

8. The method of claim 7, wherein two or more of the enzymes (a), (b), and (d) are over-expressed in the recombinant yeast host cell.

9. The method of claim 1, wherein the yeast host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, and Pichia.

10. The method of claim 9, wherein the yeast host cell is Saccharomyces cerevisiae.

11. The method of claim 6, further comprising:
(a) culturing the recombinant yeast host cell under suitable conditions to produce isobutanol from pyruvate; and
(b) recovering the isobutanol.

12. The method of claim 11, wherein said recovering is by distillation, liquid-liquid extraction, adsorption, decantation, pervaporation or combinations thereof.

13. The method of claim 11, further comprising (c) removing solids from the fermentation medium.

14. The method of claim 13, wherein said removing is by centrifugation, filtration or decantation.

15. The method of claim 13, wherein said removing step (c) occurs before said recovering step (b).