Plantas en Chimbote
SECTORIZACIÓN DE LA CIUDAD
3.1 Two approaches were used in the experiments to clone the S. cerevisiae GDHl gene. These were complementation of a gdhl mutation or heterologous probing of a S. cerevisiae gene library using a previously cloned NADP-GDH gene.Grenson et al. (1970) demonstrated that tlie GAP of S. cerevisiae
is inhibited by ammonium. However, ammonium inhibition of GAP is relieved in a gdhl mutant (Grenson and Hou 1972), resulting in sensitivity to D-amino acids in the presence of ammonium and the inability to use aUantoin (Grenson et al. 1974, and references cited therein). Strains transformed with the GDHl gene can be identified by screening transformants of a gdhl mutant for colonies which grow normally on MM supplemented with either aUantoin or D-histidine + NH4+.Comparison of the amino acid sequence of NADP-GDH from a number of prokaryotes and lower eukaryotes has shown that there is a strong sequence homology, especially in the amino-terminal region of the polypeptide (see section 1.3.1). This observation can be exploited, and a previously cloned NADP-GDH gene used to identify homologous sequences in other organisms (see Miller and Brenchley 1984, Nagasu and Hall
1985, Gxmetal. 1986).
3.1.1 To isolate the 5. cerevisiae GDHÎ gene by complementation, strain SAYl was transformed with CsCl-purified plasmid DNA from the Nasmyth gene library. Transformants were selected by using the vector-encoded LEU2 gene to complement the chromosomal marker leu2-3 ,~H2, Clones concomitantly transformed with the GDHl were screened for by replicaplating the LEU+ transformants onto MM + aUantoin, and examining the replicates for colonies exhibiting normal growth.
Transformation of protoplasts (Sherman et al. 1983) and of alkali-treated cells (Ito et al. 1983) were both tried. However, it was not possible to achieve transformation frequencies greater than 10 transformants per pg DNA with either protocol. This observation was also made using strain BC55. The basis of the refractory nature of these strains to transformation is unclear. Approximately 500 transformants were screened for GDH+ transformants. None were obtained. Due to the difficulties encountered with the transformation of gdhl strains, and the availability of suitable probes, it was decided to attempt to clone the S. cerevisiae GDHl by heterologous probing.
Approximately 5500 colonies from the Nasmyth gene library were screened with the N. crassa am gene. Cross-hybridisation was observed with 16 colonies, although the signal strengths varied. The recombinant plasmids giving the strongest signals were designated p8, p9, pi 1 and p l8, and were taken for further study.
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3.1.2 CsCl-purifîed DNA of plasmids p8, p9, p li and p l8 plus the vector YEplB were restricted with EcoRI. A Southern blot of this material was probed with the N. crassa am gene (Fig. 2). The probe hybridised to a single EcoRl fragment in each of recombinant plasmids (positions ld-4d) - this fragment contains all or part of the insert ^ig . 3). No hybridisation was observed with vector alone (position 5). It can be concluded that the probe cross-hybridises to an insert sequence which is homologous to the N. crassa am gene, and that the plasmids are not false positives due to probe-vector hybridisation
3.1.3 Although the inserts carried by the recombinant plasmids contain sequences which cross- hybridise with the N. crassa am gene, this is itself not sufficient evidence for it to be concluded that the entire GDHJ has been cloned. The simplest test is to determine if the recombinant plasmids can complement a gdhl mutation. Strain BC55 was transformed with the four plasmids, the vector YEpl3 and also the S. cerevisiae GDHl plasmid, obtained from Dr. B.D. Hall (Nagasu and Hall 1985). The growth characteristics and NADP-GDH activities of the various transformants were determined, and are presented in Tables 4 and 5.
BC55(YEpl3) grew on MM + NH4+ because, although it carries the gdhl~6 allele, glutamate was synthesised by the GS-GOGAT pathway. So the growth observed for the other transformants on ammonium was due either to the functioning of this alternative pathway or because the recombinant plasmid encoded a functional NADP-GDH. As was described in section 3.1.1, restoration of the ability of a gdhl strain to grow on aUantoin is a diagnostic test for the presence of a cloned GDHl gene. BCS5(pCYG4) can utilise aUantoin as sole nitrogen: BC55(YEpl3) cannot None of the putative GDHl
plasmids obtained in this study gave rise to transformants capable of growing on MM + aUantoin. NADP-GDH activity was measured in the recombinant strains (Table 5). Strains BC55 and BC55(YEpl3) have no detectable NADP-GDH activity, compared to 1.4U/mg for the wild type 11278b. Thus the presence of the vector does not cause restoration of wild type enzyme activities. Enzyme activity is elevated 10-fold in BC55(pCYG4) compared to %f278b. As YEpl3 is a 2ji-based plasmid, it is present in multiple copies in the ceU, so overproduction of the enzyme was expected. Strains transformed with plasmids p8, p9, pU and p l8 have enzyme activities comparable to that observed with vector alone. These recombinant plasmids do not restore wUd type NADP-GDH activity, let alone the elevated level observed with pCYG4,
The data presented in Tables 4 and 5 show that, although p8, p9, pH and p l8 may possess a sequence which cross-hybridises with the N. crassa am gene, that sequence does not specify a functional NADP- GDH.
P i g . 2 1 2 3 4 5 u d u d u d u d u d Ori — 21.2 — 5.0 — 43 — 3.5 — 2.0 — 1.6 — 1 3 —
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S o u t h e r n b l o t o f t h e f o u r p l a s m i d s c a r r y i n g t h e p u t a t i v e S . c e r e v i s i a e GDH1 g e n e . P l a s m i d DNA was r e s t r i c t e d w i t h E co R I , a n d a S o u t h e r n b l o t o f t h i s m a t e r i a l p r o b e d w i t h t h e c r a s s a am g e n e . 1 - p 8 ; 2 - p 9 ; 3 - p 1 1 ; 4 - p 1 8 ; 5 - YEp1 3 ; u - u n d i g e s t e d DNA; d - d i g e s t e d . The f i g u r e s down t h e l e f t - h a n d e d g e a r e s i z e m a r k e r s ( k b ) f r o m a H i n d l l l / E c o R I d i g e s t o fX
DNA.F ie *