Identification of the cloned S. cerevisiae LYS2 gene by an integrative transformation approach

Eibel, Hermann; Philippsen, Peter

doi:10.1007/bf00330891

Cited by 63 publications

(29 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4B). Highresolution mass spectrometry supports this finding, as the expected mass matched the mass of the chromatographically detected product peak (m/z 231.1128 [M] ϩ , expected for (31,32) and by P. chrysogenum Lys2 (2). In the latter case, a metabolic dichotomy precedes the Lys2-catalyzed step, as L-␣-aminoadipic acid also serves as the substrate for ␦-(L-␣-aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase, the gateway enzyme for penicillin biosynthesis.…”

Section: L-tyrosine Reductase A-t-sdr (Class Ii) L-α-aminoadipic Acidsupporting

confidence: 54%

Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

Kalb

Lackner

Hoffmeister

2014

Appl Environ Microbiol

View full text Add to dashboard Cite

A key step in fungal L-lysine biosynthesis is catalyzed by adenylate-forming L-␣-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP ؉ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized L-␣-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus L-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes.

show abstract

Section: L-tyrosine Reductase A-t-sdr (Class Ii) L-α-aminoadipic Acidsupporting

confidence: 54%

Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

Kalb

Lackner

Hoffmeister

2014

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Previously, ssn6lcyc8 has been mapped to a locus 4.5 centimorgans from lys2 (34). Comparison of the restriction maps of our clones and the previously cloned LYS2 locus (11) indicated that the 3' end of the LYS2 RNA maps about 2 kilobases (kb) from the right edge of the DNA segment cloned in pLN113-3.…”

Section: Resultsmentioning

confidence: 78%

Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

Schultz¹,

Carlson²

1987

Mol. Cell. Biol.

214

133

View full text Add to dashboard Cite

Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene. We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA. Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, aspecific mating defects, and failure of homozygous diploids to sporulate. In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase. An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL1O-lacZ fusion and maltase. The mating defects of MATa ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of a-factor; no deficiency of MATa2 RNA was detected. We showed that ssn6 partially restored invertase expression in a cyrl-2 mutant, although ssn6 was clearly not epistatic to cyri-2. We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). Possible functions of the SSN6 product are discussed.The SNFJ gene of Saccharomyces cerevisiae encodes a protein kinase that is required for derepression of many glucose-repressible genes, including the SUC2 (invertase) gene (5,8). Mutations in the SSN6 gene have been isolated as suppressors of the invertase derepression defect in a snfl mutant (6). The ssn6 mutations were found to cause highlevel constitutive (glucose-insensitive) invertase synthesis in both snfl and wild-type (SNFI) genetic backgrounds. These findings suggested that SSN6 is a negative regulator of SUC2 and may be a substrate of the SNFJ protein kinase. The pleiotropic phenotypes of ssn6 mutants indicated that SSN6 also affects expression of other genes; the mutants exhibited extreme clumpiness, a-specific mating defects, and a failure to sporulate as homozygous diploids (6). Genetic mapping has led to the finding that ssn6 is allelic to cyc8, a mutation that causes overproduction of iso-2-cytochrome c (34). Additional cyc8/ssn6 alleles have recently been isolated by Trumbly (45).We report here the cloning and molecular analysis of the SSN6 gene. We constructed null mutations to determine the phenotype of strains lacking SSN6 function, tested the effects of multiple copies of SSN6 on SUC2 expression, and determined the sequence of the gene. We showed that ssn6 affects expression of two other glucose-repressible genes and also explored the molecular basis of the mating defects of MATa ssn6 strains. MATERIALS AND METHODSStrains and genetic methods. The S. cerevisiae strains used in this study are listed in Table 1. The strains were constructed by standard genetic methods (39), and the genotypes of double mutants were confirmed by complementation analysis. Media and methods for scoring markers have been described (28). Yeast transformations were carried out as described prev...

show abstract

“…From the maize clones pMLl (Langridge and Feix, 1983) and pMSI (Wienand et al, 1981), containing genes for the 21 000and 19 000-dalton zein proteins, respectively, the maize inserts were transferred to the EcoRI site of the yeast integrative vector YIp35C (Eibel and Philippsen, 1983) to yield the hybrid plasmids YIpL1 and YIpSl as shown in Figure 1. This integrative vector, carrying the URA3 gene as selectable marker in Saccharomyces cerevisiae and an internal segment of the yeast L YS2 gene, was used in preference to episomal vectors to avoid problems of instability during propagation in yeast.…”

Section: Resultsmentioning

confidence: 99%

Transcription from maize storage protein gene promoters in yeast.

Langridge¹,

Eibel²,

Brown³

et al. 1984

The EMBO Journal

View full text Add to dashboard Cite

Genes coding for zein storage proteins of maize of the 19 000‐ and 21 000‐dalton size classes were integrated into chromosome II of the yeast Saccharomyces cerevisiae. Using the transformed yeast strains as an in vivo transcription system it was shown that promoter regions of the zein genes of both size classes were accurately recognized. In particular, the zein mRNA synthesis in yeast starts at the same nucleotide positions used in the maize endosperm. A difference in the promoter activity between the 21 000‐ and the 19 000‐dalton zein genes was observed.

show abstract

Identification of the cloned S. cerevisiae LYS2 gene by an integrative transformation approach

Cited by 63 publications

References 37 publications

Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

Transcription from maize storage protein gene promoters in yeast.

Contact Info

Product

Resources

About