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  • 1
    ISSN: 1432-0983
    Keywords: S. cerevisiae ; Lysine biosynthesis ; LYS5 gene ; Subclone ; Enzyme activity ; S1 analysis ; DNA homology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The LYS5 and LYS2 genes of Saccharomyces cerevisiae are required for the synthesis of α-aminoadipate reductase in the lysine pathway. The LYS5 gene, originally cloned as a DNA insert of the plasmid pSC5, has been subcloned on a 3.2 kb SphI-Sau3AI DNA fragment of the recombinant plasmid pSR7. An internal 2.1 kb HpaI-HpaI DNA fragment of the subclone, upon Southern hybridization, exhibits homology with HpaI-restricted wild-type S. cerevisiae genomic DNA. The lys5 + transformants exhibited α-aminoadipate reductase activity similar to that of wild-type cells. S1 nuclease analysis localizes the transcription initiation site relative to the detailed restriction map, and reveals the direction of transcription, as well as the transcript size of the LYS5 gene which can be no greater than 1.65 kb. From this it is estimated that the encoded polypeptide is appreciably smaller than the 4 kb LYS2 gene product. These results provide a physical and biochemical characterization of the cloned LYS5 gene. Based on these observations, it is concluded that the LYS5 gene encodes a relatively small polypeptide of the large heteropolymeric α-aminoadipate reductase.
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  • 2
    ISSN: 1432-072X
    Keywords: Fructose-1,6-bisphosphatase ; Phosphofructokinase ; Antagonistic enzymes ; Glycolytic intermediates ; ATP ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The intracellular levels of adenosine triphosphate and several glycolytic intermediates were determined in Saccharomyces cerevisiae in relation to the presence of the metabolically antagonistic enzymes phosphofructokinase and fructose-1,6-bisphosphatase. Phosphofructokinase is synthesized constitutively in cells grown in the presence of glucose and fructose-1,6-bisphosphatase derepression occurs upon the exhaustion of glucose from the growth medium. Transcriptional regulation of fructose-1,6-bisphosphatase was suggested based on experiments with wild type cells using 8-hydroxyquinoline, a known inhibitor of nuclear transcription, and with the S. cerevisiae mutant strain A364A (ts-136) blocked in the transport of nuclear RNA at non-permissive temperature. The level of phosphofructokinase was reduced more than 25-fold under conditions of high citrate accumulation in an aconitaseless, glutamate requiring mutant strain, MO-1-9B. There was a rapid decrease in the levels of adenosine triphosphate and fructose-1,6-bisphosphate at the end of log-phase of culture growth when both fructose-1,6-bisphosphatase and phosphofructokinase were present in the cells simultaneously. The changes in the levels of key glycolytic intermediates, but not the changes in adenosine triphosphate, during the simultaneous presence of these two enzymes, can be explained without involving any futile cycling.
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  • 3
    ISSN: 1432-0983
    Keywords: S. cerevisiae ; Lysine biosynthesis ; Derepression ; lys9 mutant ; Enzymes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Derepression of lysine biosynthetic enzymes of Saccharomyces cerevisiae was investigated in lys9 auxotrophs which lack saccharopine reductase activity. Five enzymes (homocitrate synthase, homoisocitrate dehydrogenase, α-aminoadipate aminotransferase, α-aminoadipate reductase and saccharopine dehydrogenase) were constitutively derepressed in all lys9 mutants with up to eight-fold higher enzyme levels than in isogenic wild-type cells. Levels of these enzymes in lys2, lys14, and lys15 S mutants were the same or lower than those in wild-type cells. The regulatory property of lys9 mutants exhibited recessiveness to the wild-type gene in heterozygous diploids. Unlike the mating type effect, homozygous diploids resulting from crosses between lys9 auxotrophs exhibited even higher levels of derepressed enzymes than the haploid mutants. Addition of a higher concentration of lysine to the growth medium resulted in reduction of enzyme levels although they were still derepressed. These results suggest that lys9 mutants represent a lesion for the saccharopine reductase and may represent a repressor mutation which in the wild-type cells simultaneously represses unlinked structural genes that encode for five of the lysine biosynthetic enzymes.
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  • 4
    ISSN: 1432-0983
    Keywords: S. pombe ; Lysine biosynthesis ; α-Aminoadipate reductase ; Enzyme regulation ; lys1 + DNA sequence ; Peptide homology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The α-aminoadipate pathway for the biosynthesis of lysine is unique to fungi. Molecular properties of the cloned lys1 + gene and the regulation of the encoded α-aminoadipate reductase (AAR) were investigated in the fission yeast Schizosaccharomyces pombe. A 5.2-kb HindIII-EcoRI fragment of S. pombe DNA, containing a functional lys1 + gene and a promoter, was subcloned to make the 10.7-kb plasmid pLYS1H. A nested 1.778-kb HindIII-EcoRI DNA fragment that complemented the lys1-131 mutant phenotype was sequenced from the plasmid pLYS1D, and shown to contain an open reading frame (ORF) of 470 amino acids, preceded by putative POLII promoter elements (TATA and CCAAT box elements, and two potential yeast GCN4-binding motifs) within 368 bp upstream of the start codon. This ORF shared with the corresponding region of the isofunctional AAR of Saccharomyces cerevisiae 49% amino-acid identity (62% similarity) overall, within which were smaller regions of marked sequence conservation. One such region coincided (95% identity) with a putative AMP-binding domain motif identified in the AAR of S. cerevisiae. In wild-type S. pombe, AAR activity from cells grown in lysine-supplemented minimal or YEPD media was less than the activity of cells grown in minimal mediu. The AAR of S. pombe was more sensitive to feedback inhibition by lysine in vitro than the AAR of S. cerevisiae. These results show the effects of extensive evolutionary divergence on the structure and expression of a pivotal enzyme in the α-aminoadipate pathway. Presumably, delineated regions of strong sequence conservation correspond to discrete domains essential to AAR function.
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  • 5
    ISSN: 1432-0983
    Keywords: Key wordsCandida albicans ; Lysine gene ; α-Aminoadipate reductase ; Regulation ; Peptide antibiotic synthetase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The unique α-aminoadipate pathway for lysine biosynthesis is present only in fungi and involves eight enzyme steps. α-Aminoadipate semialdehyde dehydrogenase, commonly called α-aminoadipate reductase (AAR), catalyzes the conversion of α-aminoadipic acid to α-aminoadipic semialdehyde by a novel mechanism. Two genes, LYS2 and LYS5, encode the heterodimeric enzyme in Saccharomyces cerevisiae. The LYS2 gene of Candida albicans was shown to be contained in the 4.8-kb insert of the plasmid pCaLYS2. This plasmid complemented lys2 mutants of both S. cerevisiae and C. albicans. The S. cerevisiae and C. albicans Lys2+ transformants exhibited 138% and 160% of wild-type AAR activity, respectively. The DNA-sequence analysis of the 4.8-kb region in plasmid pCaLYS2 and a PCR product from genomic DNA which overlapped with the 4.8-kb insert revealed a continuous ORF of 4173 nucleotides encoding 1391 amino-acid residues. The C. albicans LYS2 ORF exhibited 63.0% identity at the nucleotide level and 56.2% identity at the amino-acid level to the LYS2 gene of S. cerevisiae. The ORF is preceded by consensus sequences for the TATA-, CAAT- and GCN4-box elements. An S. cerevesiae-type transcription termination signal is seen in the 3′ flanking region. The deduced amino-acid sequence revealed a motif for an AMP-binding site and also the highly conserved core sequences common to peptide antibiotic synthetases. The LYS2 mRNA and α-aminoadipate reductase activity were repressed to a higher level in YEPD-grown cells than in cells grown in the presence of lysine or minimal medium. Additionally, AAR was shown to be feedback-inhibited by lysine and the lysine analog, thialysine. The results of the present report reveal the molecular characteristics of the LYS2 gene of C. albicans, its homology to peptide antibiotic synthetases, its divergence from the LYS2 gene of S. cerevisiae, and the regulation of AAR in C. albicans.
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  • 6
    ISSN: 1432-0983
    Keywords: S. cerevisiae ; Lysine biosynthesis ; General control ; Repression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Six of the eight enzymes of the α-aninoadipate pathway for the biosynthesis of lysine in Saccharomyces cerevisiae were examined for repressibility to lysine and for susceptibility to the general control of amino acid biosynthesis. All of the enzymes exhibited a 2 to 4 fold lower level of specific activity in the wildtype strain X2180 when grown in lysine supplemented medium as compared to minimal medium. However, levels of only three of the enzymes, α-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase, were derepressed in the leaky lysine mutant 7305d and leaky arginine mutant 7853-6c when grown in minimal medium. These observations are characteristic of enzymes under general control of amino acid biosynthesis. The remaining three enzymes, homocitrate synthease, homoaconitase and homoisocitrate dehydrogenase were repressed in 7305d cells grown in minimal or lysine supplemented medium.
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  • 7
    ISSN: 1432-0983
    Keywords: S. cerevisiae ; Lysine biosynthesis ; Cloned LYS5 gene ; Restriction map ; α-Aminoadipate reductase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In Saccharomyces cerevisiae, the functions of two unlinked genes (LYS2 and LYS5) are required for the synthesis of the lysine biosynthetic enzyme, α-aminoadipate reductase. The LYS5 gene of S. cerevisiae was cloned by functional complementation of a lys5 mutant, X4004-3A, using a YEp24 plasmid library. The cloned LYS5 gene was contained within a 7.5 kb DNA insert of the recombinant plasmid pSC5. Cloning of LYS5 gene was confirmed by second cycle transformation of a lys5 mutant with the pSC5 plasmid, growth response studies, and plasmid loss experiments with Lys5 + transformants. Analysis of restriction digests of the pSC5 plasmid revealed 3 EcoRI, 5 PvuII, 1 PstI, 1 BglII and 2 HpaI sites in the 7.5 kb insert. A 3.9 kb internal pSC5 fragment hybridized only to the plasmid pSC5, but no homology was observed with LYS2 DNA or the YEp24 vector. The pSC5 transformed Lys5 + cells and the wild-type strain exhibited same level of α-aminoadipate reductase activity, whereas lys5 mutant and plasmid-cured transformed strain exhibited none. Lys2 + transformants consistently had five times greater α-aminoadipate reductase activity when compared with the wildtype and the Lys5 + transformant. The α-aminoadipate reductase activity was repressed in lysine-grown wildtype and Lys5 + transformed cells but not in Lys2 + transformed cells. A Lys2 + and Lys5 + double transformant exhibited higher a-aminoadipate reductase activity than lys2 + or lys5 + transformant.
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  • 8
    ISSN: 1432-0983
    Keywords: S. cerevisiae ; Lysine biosynthesis ; Cloned LYS4, LYS15 genes ; Subclone, DNA homology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The plasmid pSC4 which carries a 7.8 kb yeast DNA insert at the BamHI site of the Vector YEp13, complemented simultaneously MO-59-13c lys4, LU75 1ys15 and LU32 lys4lys15 (double) mutations of Saccharomyces cerevisiae. The 1.9 kb BamHl-XbaI DNA insert of the subclone pS051 complemented the LU75 lys15 mutation. The 2.8 kb XhoI-XhoI DNA insert of the pS052 subclone, like pSC4, complemented all three mutations. The 1.9 kb BamHI-XbaI DNA and the 2.8 kb XhoI-XhoI DNA were 100 bp apart in the pSC4 DNA insert and exhibited no homology with each other upon Southern hybridization. The 1.9 kb BamHI-XbaI DNA insert exhibited homology with the pSC4 and pS051 DNA as well as the genomic DNA of MO-59-13c lys4, LU75 lys15, LU32 lys4lys15, and RC1 (LYS) when digested with appropriate restriction enzymes. The 2.8 kb Xhol-XhoI DNA insert exhibited homology with the pSC4 and pS052 DNA as well as MO-59-13c lys4, LU75 lys15, LU32 lys4lys15, and RC1 (LYS) genomic DNA, when digested with XhoI enzyme. The 2.8 kb DNA probe also hybridized with ply(A)+ RNA from RC1 and lys4 − transformant but not that from. MO-59-13c lys4 mutant.
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  • 9
    ISSN: 1434-6036
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract. We consider a model (Lengyel-Epstein) reaction-diffusion system under spatial parametric modulation and demonstrate the effect of resonance shift of the Hopf-Turing boundary. A systematic perturbative and numerical analysis shows that this shift may induce spatial inhomogeneity on an homogeneous stable state resulting in pattern formation.
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  • 10
    ISSN: 1434-6036
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract The anomalous dimension index η assumes its critical value for small momenta and vanishes for large momenta. We provide, in the form of a quadrature, a momentum dependent η(k) which interpolates between the above limits. High- and low-momentum expansions for the spectral function are obtained, leading to a Padé approximant from which η(k) is determined, thereby giving a crossover critical correlation function.
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