Patent Application: US-26449294-A

Abstract:
described are methods and means for the construction of strains of yeast capable of producing cellulolytic enzymes . this is achieved by the transfer of chromosomal genes or cdna copies of mrnas coding for cellulolytic enzymes isolated from the fungus trichoderma reesei to yeast cells using recombinant dna vectors capable of replicating in yeast . the correct expression of these cellulolytic genes in yeast leads to the production of cellulolytic enzymes which are secreted from the cell . this allows the yeast to hydrolyze 3 - 1 , 4 - glucan substrates such as cellulose .

Description:
the definitions used in this detailed description are as defined in the gilbert and talmadge u . s . pat . no . 4 , 338 , 397 . t . reesei strain vtt - d - 80133 , a mutant strain with improved production of cellulolytic enzymes derived from qn 9414 ( 17 ) after several successive mutation steps ( 18 ), was used for isolation of the genes from cellobiohydrolase i ( cbh i ), cellobiohydrolase ii ( cbh ii ) and endoglucanase ii ( ehdo ii ), now known as ednoglucause i ( egi ). escherichia coli strains q358 and q359 and the phage λ1059 , used in the construction of the t . reesei gene bank were provided by dr . j . karn ( 19 ). e . coli hb 101 was used as a host in 5 transformations with the plasmid pbr 322 . e . coli jm 101 and the phage m 13 mp 7 ( 20 ) and the plasmids puc 8 and puc 9 ( 21 ), used in the dideoxy sequencing , were from the laboratory of f . sanger . yeast strains used were saccharomyces cerevisiae oll ( mata leu 2 - 3 leu 2 - 112 his 3 - 11 his 3 - 15 ura 3 - 251 ura 3 - 373 ) ( 22 ) and s . cerevisiae mt302 - 1c ( mata arg 5 - 6 leu 2 - 3 leu 2 - 112 his 3 - 11 his 3 - 15 pep 4 - 3 ade 1 ) ( 23 ). a 12 kb cosmid p3030 obtained from barbara hohn , which replicates both in e . coli and in yeast was used as vector for transferring the chromosomal copy of cbh i to yeast . cosmid p3030 contains genes for ampicillin and tetracycline resistance in e . coli and the his3 gene for selection in yeast . the vector contains a cos site which enables it to be packaged into infective λ phage particles in vitro and the yeast 2 μecod fragment . yeast expression vector containing the phosphoglycerokinase ( pgk ) gene promoter was used for expression of the cdna copies of cellulase genes in yeast ( 23 ). restriction enzymes were purchased from amersham ( uk ), boehringer mannhelm ( fdr ) and bethesda research laboratories ( gaithersburg , md .) and used according to the manufacturers &# 39 ; instructions . t4 ligase and the dna polymerase i large subunit were from biolabs and the calf intestine phosphatase from boehringer mannhelm . reverse transcriptase was from dr . j . w . beard ( life sciences inc ., st . petersburg , fla .). protoplasting enzyme , zymolyase 60000 was obtained from kirin brewery co ., japan . klenow fragment of e . coli polymerase i was from boehringer mannheim . e . coli hb101 was grown in l - broth . transformants were selected on l - plates supplemented with 1 . 5 % agar and containing 100 μg / ml ampicillin . the concentration of tetracycline added to l - plates was 10 μg / ml . complete medium ypg for growth of yeast contained 1 % yeast extract , 2 % peptone , and 2 % glucose . yeast minimal medium , ymb , contained 0 . 67 % yeast nitrogen base ( difco , detroit , usa ) and 2 % sugar ( lactose , cellobiose , starch or glucose ). the final concentration of amino acids added was as described ( 24 ). the solidifying agent on yeast plates was 2 % agar ( difco bacto agar ). in yeast protoplast plating medium 1 . 2m sorbitol was added as an osmotic stabilizer . the top agar used in plating the yeast protoplasts for regeneration was prepared as minimal medium but using 3 % purified agar ( difco ) as a solidifying agent . all methods unless otherwise specified are as described in maniatis et al . 1982 ( 25 ). isolation and characterization of the cellulolytic genes from the fungus t . reesei polyadenylated ( polya + ) messenger rna isolated from t . reesei mycelia actively producing celluloses directs in the in vitro synthesis -- in a rabbit reticulocyte lysate -- of a number of large polypeptides that are precipitated by antibody prepared against purified cellulolytic enzymes . messenger rna isolated from repressed glucose grown mycelia does not direct the synthesis of these cellulase - specific polypeptides . this difference between induced and repressed populations was used to identify a collection of hybrid phages containing t . reesei genes strongly expressed during production of cellulolytic enzymes . for the isolation of cellulase - specific , induced mrnas t . reesei ( strain vtt - d - 80133 ) was grown as described by bailey and nevalainen ( 26 ) except that the medium contained 2 % lactose and 2 % of a soluble extract of distillers spent grain . samples taken during cultivation were assayed for activity against dyed avicel , hydroxyethylcellulose ( hec ) and for soluble protein ( 26 ). estimation of reducing sugars was by the method of sumner ( 27 ). cellular rna from mycelia was isolated by a modification of the method of ohi and short ( 28 ). the frozen mycelia was ground to a fine powder under liquid nitrogen and suspended in a buffer containing 20 mm tris - hcl ( ph 7 . 6 ), 0 . 1m nh 4 1 mm mg ( oac ) 2 , 10 mm na - iodoacetate , 0 . 5 mg / ml polyvinylsulfate and 2 % na - dodecyl sulfate ( sds ). following incubation at 37 ° c . for 30 minutes , insoluble material was removed by centrifugation at 13000 g for 10 minutes . the poly ( a ) + fraction was purified by chromatography through an oligo ( dt ) cellulose column ( bethesda research laboratories ( 29 ) and in vitro translation was carried out with a rabbit reticulocyte lysate using 35 s - methionine ( amersham international ltd ) ( 30 ). immunoprecipitation was carried out according to dobberstein ( 31 ) using antiserum prepared against purified cbh i , cbh ii or endo ii ( now egi ), or with the corresponding preimmune serum . table 1 shows the molecular weights of proteins precipitated by antiserum against specific cellulases analysed on 7 . 5 - 15 % sds polyacrylamide gels ( 32 ). table 1______________________________________antiserum in vivo in vitro______________________________________cbh i 71 000 67 000cbh ii 63 000 48 000endo ii ( now egi ) 62 000 53 000______________________________________ the construction of the t . reesei gene bank was carried out as follows . conidia of trichoderma reesei were germinated in a liquid medium containing 1 . 5 % kh 2 po 4 , 0 . 5 % ( nh 4 ) 2 so 4 , 0 . 06 % mgso 4 · 7h 2 o , 0 . 06 % cacl 2 , 0 . 15 % proteose peptone , 0 . 03 % urea , 2 % sucrose and minimal salts . cultures were incubated with shaking at 29 ° c . for about 12 h . the isolation of nuclei was carried out using a slightly modified method of hautala et al . ( 33 ). dna was isolated from a crude nuclear pellet obtained by differential centrifugation of homogenized mycelium . the crude nuclear pellet was treated with sds - amylase solution ( 100 mm edta ph 8 . 0 , 140 mm nacl , 1 % nadecylsulfate and 3 . 3 % α - amylase obtained from merck , darmstadt , frg ) for 1 h at 37 ° c . proteinase k ( final concentration 0 . 8 % w / v ) was then added and incubation was continued for 2 h at 37 ° c . with gentle shaking . after incubation , cell debris was removed by centrifugation and dna was precipitated from the supernatant with ethanol . the dna was then purified by cscl centrifugation . the chromosomal dna from t . reesei was partially digested with mboi and sized by sucrose density gradient centrifugation . fifteen - 20 kb fragments were ligated to bam hi - cleaved x 1050 dna . in vitro packaging of the recombinant molecules was carried out using packaging extracts prepared by the method of hohn as described by maniatis et al . ( 25 ). recombinant phages were transferred from the agar to nitrocellulose filters ( schleicher & amp ; schull , ba 85 ) as described by benton and davis ( 34 ). cdnas made from induced mrna ( described earlier ) and from mrna isolated from fungus grown in the presence of glucose were used as probes . cdna first strand synthesis was carried out by the procedure of efstradiatis et al . ( 35 ) but using 10 μci of 32 pαatp per 50 μl reaction . the in situ plaque hybridization was carried out according to maniatis et al . ( 25 ). hybridization was detected by autoradiography of the filters on kodak x - omat film . positive plaques were picked into 1 ml of sm ( 25 ) and a drop of chloroform and stored at - 4 ° c . hybrid phage hybridizing only to cdna made with induced mrna containing cellulase coding sequences were purified extensively and retested by hybridization to both probes . a number of different hybrid clones that hybridized strongly to the induced cellulase probe were identified and selected for further analysis . the hybrid phages containing genes induced when the fungus produces cellulases were first grouped according to their restriction enzyme patterns . then the particular cellulase gene in each group was identified by hybrid selection of messenger rna . dbm paper was obtained from schleicher and schull ( keene , n . h .) and activated according to the maker &# 39 ; s instructions . binding of dna to the activated paper and rna hybridization and elution was carried out according to maniatis et al . ( 25 ). rna was translated with a rabbit reticulocyte lysate supplied by amersham international ltd . and the proteins produced were labeled with 35 s - methionine . the proteins were analysed by autoradiography on kodak x - omat film after separation on a 7 - 15 % polyacrylamide gradient denaturing gel . the size of the proteins obtained from particular phage by hybrid selection and their cross reaction with specific antiserum is shown in table 2 . table 2______________________________________hybrid phage no . 44a w17a w12a______________________________________mol . weight of major protein 67 000 48 000 53 000produced from hybrid selectedmessagecross reaction of major proteinwith antisera againstcbh i + - - cbh ii - + - endo ii ( now egi ) - - + ______________________________________ single and double digests of the clone 44a , were analyzed on 0 . 6 % and 1 . 5 % agarose gels . the fragments were electrophoretically transferred to gene screen membranes ( new england nuclear , ma ) and hybridized to the induced cdna probe as instructed by the manufacturer . this procedure permitted the construction of restriction enzyme maps of the three cellulose genes . these restrictions enzyme maps are shown in fig1 and 3 . the nucleotide sequence of the cbh i , cbh ii and endo ii ( now egi ) genes was generated by dideoxy sequencing ( 36 ) using restriction enzyme fragments or dna fragments obtained by the &# 34 ; shotgun &# 34 ; procedure ( 37 ). the construction of a yeast vector containing the cbh i chromosomal gene the hybrid phage 44a ( 11 ) dna containing the cbh i of trichoderma reesei hypercellulolytic mutant strain vtt - d - 80133 , was digested with pst i to give a mixture of fragments one of which being about 12 kb and containing the entire cbh i gene with its own regulatory sequences . the resulting dna - fragments were ligated with the yeast cosmid p3030 digested partially with the same enzyme . the yeast strain oll was transformed to his + with the dna - mixture described above . transformation was carried out essentially as described by gerbaud et al . ( 38 ). transformed cells were plated on yeast minimal medium with leucine and uracil but lacking histidine . the clones were further tested in situ plague hybridization for the presence of the cbh i gene originated from t . reesei . the presence of an intact cbh i gene in yeast was ensured by isolating total dna ( 39 ) from a transformant colony and digesting it with restriction enzymes bgl ii and hinc ii . dna was transferred to nitrocellulose filter ( 40 ) from agarose gel and hybridized to a 1 . 113 probe ( 41 ) containing the 0 . 7 kb eco ri fragment from cbh i gene . fig4 shows the construction of a hybrid plasmid containing the cbh i gene . isolation of full length cdnas coding for the enzymes cbh i , cbh ii and endo ii ( now egi ) a cdna bank from t . reesei was made from induced mrna isolated from cells as described earlier . however , after the frozen mycelia had been ground under liquid nitrogen it was suspended in 5 volumes a guanidinium isothiocyanate buffer as described by maniatis et al . ( 25 ). the rna preparation was then carried out as described ( 42 ). cdna first strand synthesis was carried out according to maniatis ( 25 ) and the second strand was carried out according to gubler and hoffmann ( 43 ). the double stranded cdna was then treated with t 4 - polymerase to give blunt ends and small cdnas less than 500 nucleotides long removed by passage through a cl - 4b column ( pharmacia ). long cdnas were then ligated to a sma i digested and phosphatase treated preparation of puc 8 vector . the ligation mixture was used to transform e . coli strain jm 105 and the cdna bank was stored on nitrocellulose filters . full length cdnas coding for cbh i , cbh ii and endo ii ( now egi ) were isolated from a cdna bank using specific restriction fragments as probes . for the identification of ceh i , a radioactive eco ri - hind iii fragment from the 5 &# 39 ; end of the chromosomal gene was used to identify long cdnas . a plasmid ptt01 from a clone containing sequences homologous to this eco ri - hind iii fragment was further characterized by sequencing of the cdna ends by double stranded dideoxy sequencing . 1 μg of purified plasmid was denatured in 0 . 4m naoh at room temperature for 5 minutes at a concentration of 100 ng / μl . 5 μl of sequencing or reverse sequencing primer ( amersham ) was added and the mixture was precipitated with ethanol . after washing the pellet was resuspended in 10 μl at 14 mm tris ph 8 - 7 mm mgcl 2 . sequencing reactions were done according to general methods ( 36 ) except that temperature was kept at 37 ° c . cbh ii cdnas were isolated using a pvu ii fragment from the 5 &# 39 ; end of the chromosomal gene and the plasmid ptt09 characterized as for the cbh i cdna . endo ii ( now egi ) cdnas were identified using a kpn i - sal i fragment from the 5 &# 39 ; end of the gene and plasmid ptt11 also characterized as for the cbh i cdna . plasmid ptt11 is shown on fig9 . a transformed e . coli host ( alko2296 ) carrying plasmid ptt11 ( called pttc11 ) was deposited at the dsm - deutsche sammlung von mikroorganismen und zellkulturen gmbh on jan . 15 , 1992 as deposit number dsm6873 . all cdnas were then sequenced to determine that their sequence corresponded to that of the gene from which they are transcribed . the dna sequences of cbh ii and endo ii ( now egi ) cdnas are shown in fig5 and 6 . the cdna sequence of cbh i was identical to that already described ( 10 ). the construction of expression vectors containing cdnas for the production of fungal cellulases in yeast the efficient yeast expression vector pma 91 has been assembled using the regulatory sequences of the yeast phosphoglycerokinase ( pgk ) gene ( 23 ). the sequences coding for the amino acid sequence of the enzyme have been removed from the gene and replaced by a single bgl ii site . this deleted gene has then been inserted into a yeast / coli shuttle plasmid . the cbh i cdna was removed from plasmid ( ptt01 fig7 ) by digestion with hinc ii and the cdna fragment isolated from an agarose gel . pma 91 , the expression vector was cleaved with bgl ii and the ends were filled in with the klenow fragment . the vector was treated with phosphatase , ligated to the cdna and transformed into e . coli strain hb101 by selection for expression of the vector leucine gene ( fig7 ). plasmid dna was isolated from a number of transformants and those clones containing the cdna insert in the correct orientation with respect to the pgk pro - motor -- as identified by restriction enzyme analysis -- were retained . dna from one of these clones ( pmp11 ) was then transformed into yeast strain mt 302 - 1c by the method described earlier by selection of the leucine marker of pma 91 resulting in strain vtt - rc - 84011 . cbh ii cdna was removed from plasmid ptt 09 using eco ri and bam hi . the ends of the dna were filled in with klenow fragment . the cdna fragment was then isolated from an agarose gel and ligated to the vector pma 91 prepared as for cbh i . the ligation mix was transferred into hb101 and clones containing the cdna in the correct orientation identified . fig8 shows the dna sequence at the junctions between pma 91 and the cdna . plasmid pmp 29 with the cdna in the correct orientation was then used to transform yeast mt302 - 1c by selection for the leucine marker to give strain vtt - rc - 84012 . the endo ii ( now egi ) cdna was tranferred to pma 91 exactly in the same way as cbh ii cdna . fig9 shows the dna sequences at the junctions between pma 91 and the endo ii ( now egi ) cdna . the plasmid , pmp 311 containing the endo ii ( now egi ) cdna in the correct orientation was transferred to yeast as described earlier to give strain vtt - rc - 84013 . culturing the hybrid yeast strains to produce the cellulolytic enzymes , cbh i , cbh ii and endo ii ( now egi ) strain vtt - rc - 84001 containing yepnp03 was grown in a yeast minimal medium with leucine and uracil for three days after which complete medium ( 1 / 3 volume ) was then added to allow the cells to pass through one more division . strains vtt - rc - 84011 ( cbh i cdna ), vtt - rc - 84012 ( cbh ii cdna ) and vtt - rc - 84013 ( endo ii ( now egi ) cdna ) were grown in a yeast minimal medium containing arginine , histidine and adenine for three days after which complete medium 1 / 3 volume was added to allow the cells to pass through one more division . the final volume of the cultures was about 150 ml . three fractions were prepared from hybrid yeast cultures for analysis of enzyme activity . fraction 1 comprised the growth medium without the cells . fraction 2 comprises the supernatant left when protoplasts are pelleted and fraction 3 comprises the supernatant of lysed protoplasts . after cultivation yeast cells were collected by centrifugation and the supernatant was saved ( fraction 1 ). the resulting pellet was washed twice with distilled water and 1 . 2m sorbitol . the pellet was then resuspended in protoplasting buffer ( 1 . 2m sorbitol , 10 mm tris and 10 mm cacl , ph 7 . 6 ) and zymolyase 60000 was added at a concentration of 30 μg / ml of protoplasting suspension . suspension was incubated in a waterbath at 37 ° c . for 60 minutes with gentle shaking . the protoplasts so formed were pelleted and the resulting supernatant ( periplasmic cell contents ) ( fraction 2 ) saved for enzyme activity determinations . in some cases fractions 1 and 2 were concentrated by ultrafiltration ( amicon ). protoplast pellets were washed with cold 1 . 2m sorbitol and resuspended in 1 . 2 ml of 5 mm citrate buffer ph 5 . 0 , pelleted and the supernatant was saved ( fraction 3 ). the three different fractions were tested for cbh i enzyme activity using amorphous ball milled cellulose which is attached only by cellobiohydrolases ( 44 ). the total protein concentration of the samples was about 300 μg / ml . hydrolysis of the substrate caused by active cellobiohydrolase enzyme was measured by following the change in absorbance at 620 nm . cbh i type activity was found only in fraction 2 , the periplasmic or intramural space . fig1 shows the activity of the cbh i enzyme produced by the yeast strain vtt - rc - 84001 and secreted into the intramural space as compared to the control yeast containing only vector p3030 dna and 1 μg of trichoderma cbh i . this figure shows that the hybrid yeast strain produces active cbh i which appears to be at least as resistant to incubation at 50 ° c . for 3 days as in the native enzyme . the cbh i produced by the yeast represents 1 - 2 % of the protein of intramural space protein . as the intron sequences of fungus are different from those of yeast it is not likely that yeast would process off the fungus gene introns . probably for that reason the product of the chromosomal gene remains in the yeast periplasmic space and is not secreted from the cell as is the product coded by the cdna sequence . this result suggests that transferring the chromosomal gene coding for cbh i to yeast , results in the production of a smaller protein , which , however , has the same type of activity as the full length cellulase . the three different fractions were tested for cbh i enzyme activity as just described . however , in this case , most of the cbh i type activity was found in the growth medium . the results , with a final protein concentration during hydrolysis of 5 μg / ml is very similar to that shown in fig1 . the cbh i enzyme produced with this construction represented 1 - 5 % of total cell protein . the three different fractions were tested from cellobiohydrolase activity as described for strain vtt - rc - 84001 . as with strain vtt - rc - 84011 , most of the cellobiohydrolase type activity was found in the growth medium . the results with a final protein concentration of 10 μg / ml is similar to that shown in fig1 . the cbh ii enzyme produced with this construction represented 1 - 5 % of total cell protein . the three different fractions were tested for endoglucanase activity by following the hydrolysis of 0 . 1 % β - glucan at 50 ° c . the reducing sugars liberated in 5 minutes ( overnight ) were measured as glucose using the dinitro salisylic acid method ( 45 ). most of the endo ii ( now egi ) activity was found secreted into the growth medium . the endo ii ( now egi ) enzyme produced with this construction represented 1 - 5 % of total cell protein . it is considered that the invention and many of its attendant advantages will be understood from the foregoing description and that it will be apparent that various changes may be made in the steps of the described method for mature protein synthesis without departing from - 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