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F K Zimmermann

Publications and source records attributed to F K Zimmermann.

At least 37 records · Page 2Linked to original sources
PubMed1994

Characterization of the essential yeast gene encoding N-acetylglucosamine-phosphate mutase.

A previously cloned gene of Saccharomyces cerevisiae, which complements the growth defect of a phosphoglucomutase (pgm1 delta/pgm2 delta) double deletion mutant on a pure galactose medium [Boles, E., Liebetrau, W., Hofmann, M. & Zimmermann, F. K. (1994) Eur. J. Biochem. 220, 83-96], was identified as the structural gene encoding N-acetylglucosamine-phosphate mutase. The complete nucleotide sequence of the gene, AGM1, and surrounding regions were determined. AGM1 codes for a predicted 62-kDa protein with 557 amino acids and is located on chromosome V adjacent to the known gene PRB1 encoding protease B. No extended nucleotide or amino acid sequence similarities could be found in the databases, except for a small region of amino acids with high similarity to the active-site consensus sequence of hexosephosphate mutases. Three putative pheromone-responsive elements have been identified in the upstream region of the AGM1 gene. The gene is essential for cell viability. An agm1 deletion mutant progresses through only approximately five cell cycles to form a 'string' of undivided cells with an abnormal cell morphology resembling glucosamine auxotrophic mutants. Expression of the AGM1 gene on a multi-copy plasmid led to a significantly increased N-acetylglucosamine-phosphate mutase activity. Unlike over-expression of the AGM1 gene in a pgm1/pgm2 double deletion mutant which could restore phosphoglucomutase activity, over-expression of the PGM2 gene encoding the major isoenzyme of phosphoglucomutase did not increase N-acetylglucosamine-phosphate-mutase activity and did not restore growth of agm1 deletion mutant cells. Our observations indicate that the different hexosephosphate mutases of S. cerevisiae have partially overlapping substrate specificities but, nevertheless, distinct physiological functions.

Amino Acid Sequence↗
PubMed1994

A family of hexosephosphate mutases in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae PGM1 and PGM2 genes encoding two phosphoglucomutase isoenzymes have been isolated and sequenced. The derived protein sequences are closely related to one another and show distinct sequence similarities to the human and rabbit phosphoglucomutases, especially in the region supposed to constitute the active site. PGM1 and PGM2 are located on chromosomes XI and XIII, respectively, just upstream of the known genes YPK1 and YKR2 coding for a pair of closely related putative protein kinases. These observations suggest that an extended region of DNA arose by the process of gene duplication. Cells deleted for both, PGM1 and PGM2, could not grow on galactose. No residual phosphoglucomutase activity could be measured in crude extracts or in permeabilized cells of pgm1/2 double mutants. Unexpectedly, growth with glucose was not impaired and the mutant cells were still able to accumulate trehalose and glycogen, although at a reduced level. Two further genes could be isolated and characterized which when over-expressed on a multi-copy plasmid could restore growth on galactose of the pgm1/2 double deletion mutant. Multi-copy complementation was due to a sharply increased level of phosphoglucomutase activity. Partial sequencing and characterization of the two genes revealed one of them to be SEC53 encoding phosphomannomutase. No extended sequence similarities could be found in the databases for the second gene. However, part of the derived amino acid sequence contained a region of high similarity to the active-site consensus sequence of hexosephosphate mutases from different organism. Further investigations suggest that a complex network of mutases exist in yeast which interact and can partially substitute for each other.

Amino Acid Sequence↗
PubMed1994

Sequence and function analysis of a 9.46 kb fragment of Saccharomyces cerevisiae chromosome X.

In the framework of the European yeast genome sequencing project, we have determined the nucleotide sequence of the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 9464 base pairs of this cosmid located on the left arm of Saccharomyces cerevisiae chromosome X. This sequence contains two new open reading frames and includes the published sequences of the RADH gene (also identified as SRS2/HPR5) and the 3'-end of the gene BCK1/SLK1/SSP31. Deletion mutants of the two unknown genes J0909 and J0911 are viable.

Amino Acid Sequence↗
PubMed1994

Sequence and function analysis of a 9.74 kb fragment of Saccharomyces cerevisiae chromosome X including the BCK1 gene.

In the framework of the European BIOTECH project for sequencing the Saccharomyces cerevisiae genome, we have determined the nucleotide sequence of the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 9743 base pairs of sequence derived from the left arm of chromosome X. This sequence reveals three new open reading frames and includes the published sequence (5' end and open reading frame) of the gene BCK1/SLK1/SSP31 also identified as ORFAA. Deletion mutants of two earlier unknown open reading frames J0840 and J0904 are viable and the open reading frame J0902 is essential for yeast growth.

Amino Acid Sequence↗
PubMed1993

The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant.

Phosphoglucose isomerase pgi1-deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose. These growth defects could be suppressed by an over-expression on a multi-copy plasmid of the structural gene GDH2 coding for the NAD-dependent glutamate dehydrogenase. GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD-glutamate dehydrogenase. Suppression of the pgi1 deletion by over-expression of GDH2 was abolished in strains with a deletion of the glucose-6-phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH-dependent glutamate dehydrogenase. Moreover, this suppression required functional mitochondria. It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway. Over-expression of the NAD-dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2-oxoglutarate which can be converted back to glutamate by the NADPH-dependent glutamate dehydrogenase with the consumption of NADPH. Consequently, over-expression of the NAD-dependent glutamate dehydrogenase causes a substrate cycling between 2-oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria. Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH.

Amino Acid Sequence↗
PubMed1993

TKL2, a second transketolase gene of Saccharomyces cerevisiae. Cloning, sequence and deletion analysis of the gene.

Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identity with TKL1 [Sundström, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.

Amino Acid Sequence↗
PubMed1993

Sequence of a 4.8 kb fragment of Saccharomyces cerevisiae chromosome II including three essential open reading frames.

The nucleotide sequence of a fragment of 4867 base pairs of Saccharomyces cerevisiae chromosome II has been determined. The sequence contains three complete open reading frames. In addition to the already known gene RPB5, coding for a subunit shared by all three DNA directed RNA polymerases, two new open reading frames could be identified. YBR12.03 codes for a protein of 183 amino acids with homology to one of the proteins of the Bacillus subtilis riboflavin biosynthesis operon (RibG). Deletion mutants of YBR12.03 can germinate but stop growing after five to seven cell divisions on YPD. Supplementation with high concentrations of riboflavin does promote growth. YBR12.05 codes for a protein of 386 amino acids with homology to STI1, a stress-inducible protein of S. cerevisiae. Deletion mutants of YBR12.05 are not viable.

Amino Acid Sequence↗
PubMed1993

Different signals control the activation of glycolysis in the yeast Saccharomyces cerevisiae.

The glycolytic pathway in Saccharomyces cerevisiae is activated by fermentable sugars at several steps. Mutants with deletions of genes coding for enzymes of the upper part of glycolysis were used to characterize the triggering mechanisms. Synthesis of fructose-2,6-bisphophate is catalysed by two 6-phosphofructo-2-kinase isoenzymes, one of which is activated by fermentable sugars while synthesis of the second enzyme is induced (Kretschmer and Fraenkel, 1991). Increase in the level of fructose-2,6-bisphosphate is demonstrated to depend on an internal metabolite upstream of the phosphoglucose isomerase reaction. The signalling process correlates with distinct temporal changes in the concentration of glucose-6-phosphate but not with its absolute level, indicating an adaptational mechanism. It is independent of the uptake and phosphorylation systems used by different sugars. Interestingly, this increase, although delayed, could also be observed in strains lacking the rapid cAMP increase after sugar addition which is thought to be responsible for the activating process. Synthesis of glucose-6-P and fructose-6-P is needed for the complete induction of pyruvate kinase and inactivation of fructose-1,6-bisphosphatase. On the other hand, induction of pyruvate decarboxylase depends mainly on a signal in the lower part of glycolysis.

Carbohydrate Metabolism↗
PubMed1993

Sequence and function analysis of a 4.3 kb fragment of Saccharomyces cerevisiae chromosome II including three open reading frames.

The nucleotide sequence of a fragment of 4337 base pairs of Saccharomyces cerevisiae chromosome II has been determined. The sequence contains three open reading frames, one of them being incomplete. Deletion analysis showed that YBR12.31 is essential for yeast growth, while deletion mutants of YBR12.32 and YBR12.33 are viable. YBR12.33 is identical to SMY2, isolated as a suppressor of a myo2 mutant (Lillie, S.H. and Brown, S.S., unpublished, EMBL M90654).

Amino Acid Sequence↗
PubMed1993

Lysine144 is essential for the catalytic activity of Saccharomyces cerevisiae transaldolase.

Replacement of lysine144 by glutamine in the pentose phosphate pathway enzyme transaldolase of Saccharomyces cerevisiae is associated with the complete loss of activity indicating the essential role in catalysis. Neither histidine nor cysteine is important for catalytic activity as proposed for the Candida utilis enzyme. Also we could not find any evidence for a half-site character of the enzyme as described for transaldolase of C. utilis. Therefore, the reaction mechanisms for the two enzymes are different.

Amino Acid Sequence↗
PubMed1993

Induction of pyruvate decarboxylase in glycolysis mutants of Saccharomyces cerevisiae correlates with the concentrations of three-carbon glycolytic metabolites.

Pyruvate decarboxylase, PDCase, activity in wild-type yeast cells growing on ethanol is quite low but increases up to tenfold upon addition of glucose, less with galactose and only slightly with glycerol. PDCase levels in glycolysis mutant strains growing on ethanol or acetate were higher than in the wild-type strain. These levels correlated with the sum of the concentrations of three-carbon glycolytic metabolites. The highest accumulation was observed in a fructose bisphosphate aldolase deletion mutant concomitant with the highest PDCase activity ever observed under gluconeogenic conditions. Glucose addition induced an increase in PDCase activity in all mutants. However, the enzyme activities never reached wild-type level. On the other hand, the PDCase levels in the different mutants again correlated with the sum of the concentrations of the three-carbon glycolytic metabolites. This was interpreted to mean that full induction of PDCase activity requires the accumulation of hexose- and triosephosphates.

Carbon↗
PubMed1993

Saccharomyces cerevisiae phosphoglucose isomerase and fructose bisphosphate aldolase can be replaced functionally by the corresponding enzymes of Escherichia coli and Drosophila melanogaster.

Two glycolytic enzymes, phosphoglucose isomerase and fructose-1,6-bisphosphate aldolase, of Saccharomyces cerevisiae could be replaced by their heterologous counterparts from Escherichia coli and Drosophila melanogaster. Both heterologous enzymes, which show respectively little and no sequence homology to the corresponding yeast enzymes, fully restored wild-type properties when their genes were expressed in yeast deletion mutants. This result does not support notions of an obligatory formation of glycolytic multi-enzyme aggregates in yeast; nor does it support possible regulatory functions of yeast phosphoglucose isomerase.

Animals↗
PubMed1993

Pentose-phosphate pathway in Saccharomyces cerevisiae: analysis of deletion mutants for transketolase, transaldolase, and glucose 6-phosphate dehydrogenase.

Deletion mutants for the yeast transketolase gene TKL1 were constructed by gene replacement. Transketolase activity was below the level of detection in mutant crude extracts. Transketolase protein could be detected as a single protein band of the expected size by Western-blot analysis in wild-type strains but not in the deletion mutant. Deletion of TKL1 led to a reduced but distinct growth in synthetic medium without an aromatic amino-acid supplement. We also isolated double and triple mutants for transketolase (tkl1), transaldolase (tal1), and glucose 6-phosphate dehydrogenase (zwf1) by crossing the different mutants. A tal1 tkl1 double mutant grew nearly like wild-type in rich medium. Only the tkl1 zwf1 double and the tal1 tkl1 zwf1 triple mutant grew more slowly than the wild-type in rich medium. This growth defect could be partly alleviated by the addition of xylulose but not ribose. The triple mutant still grew slowly on a synthetic mineral salts medium without a supplement of aromatic amino acids. This suggests the existence of an alternative but limited source of pentose phosphates and erythrose 4-phosphate in the tkl1 zwf1 double mutants. Hybridization with low stringency showed the existence of a sequence with homology to transketolase, possibly a second gene.

Base Sequence↗
PubMed1992

Tests for recombinagens in fungi.

Three types of mitotic recombination can be studied in Aspergillus nidulans and Saccharomyces cerevisiae: (1) The classical type of reciprocal mitotic crossing-over which can be detected when it occurs between non-sister chromatids at the four-strand stage followed by co-segregation of a crossing-over and a non-crossing-over chromatid in the subsequent mitotic division. Consequently, mitotic crossing-over reflects cellular responses to primary genetic damage in the G2 phase of the cell cycle. (2) Mitotic gene conversion is a unidirectional event of a localized transfer of genetic information between non-sister chromatids which in yeast can extend to segments of up to 18 cM and even beyond 22 cM in Aspergillus nidulans. Mitotic gene conversion can also occur between unreplicated chromatids and lead to the expression of the newly created genotype without any need for a subsequent mitotic cell division. It reflects a cellular response in G1. (3) Mitotic sister-strand gene conversion can be studied in a recently constructed strain with the same technical ease as classical non-sister chromatid gene conversion. It can be induced by chemicals which do not induce mutation in the Salmonella system and non-sister chromatid gene conversion. Mitotic segregation in Saccharomyces cerevisiae results almost exclusively from crossing-over and gene conversion whereas mitotic chromosomal malsegregation contributes only very little. In contrast to this, in Aspergillus nidulans, both processes contribute considerably so that mitotic segregants always have to be tested for their mechanistic origin.

Aspergillus nidulans↗
PubMed1992

Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione, 2,3-hexanedione, ethyl acrylate, dibromoacetonitrile and 2-hydroxypropionitrile induce chromosome loss in Saccharomyces cerevisiae.

Induction of mitotic chromosome loss could be demonstrated for the dialdehyde glyoxal, the diketones 2,3-butanedione and 2,3-hexanedione, ethyl and methyl carbamate, ethyl acrylate, dibromoacetonitrile, 2-hydroxypropionitrile and formaldehyde, but only when they were combined with subacute concentrations of propionitrile, which is a strong inducer of chromosomal malsegregation. The same chemicals did not induce mitotic chromosome loss when applied in pure form. However, glyoxal, ethyl acrylate, dibromoacetonitrile and formaldehyde when applied in pure form also induced mitotic recombination. Respiratory deficiency was induced, in the absence of propionitrile, by these recombinogenic agents and also by 2,3-hexanedione and 2-hydroxypropionitrile which are not recombinogenic.

Acetonitriles↗
PubMed1992

Glycogen metabolism in a Saccharomyces cerevisiae phosphoglucose isomerase (pgil) disruption mutant.

Disruption of the gene pgil of Saccharomyces cerevisiae, which codes for phosphoglucose isomerase, results in a dramatic increase in the amount of intracellular glycogen in early exponential cultures. The level of glucose 6-phosphate was much higher in mutant than in wild-type cells. Phosphorylase a activity and the state of activation of glycogen synthase were also investigated. Phosphorylase a activity was rather low along the culture in wild-type cells, whereas it was consistently higher in mutants. Glycogen synthase was mostly in the active form in early-medium exponential cultures in wild-type cells whereas the activation state of this enzyme in mutant cells, although lower at the earlier steps of the culture, did not differ from wild-type cells at later stages. The fact that the intracellular levels of UDP-glucose are markedly increased in mutant cells suggest that the observed accumulation of glycogen results from a rise in substrate availability rather than from the activation of the enzyme responsible for the synthesis of the polysaccharide.

Enzyme Activation↗