Isolation and molecular analysis of the phosphoglucose isomerase structural gene of Saccharomyces cerevisiae

Summary The PGI1 gene of Saccharomyces cerevisiae coding for the glycolytic enzyme phosphoglucose isomerase has been cloned by complementation of a mutant strain (pgi1) with a strongly reduced phosphoglucose isomerase activity. A genomic library constructed in the yeast multicopy vector YEp13 (Nasmyth and Tatchell 1980) was used. Four plasmids containing an overlapping region of 4.1 kb were isolated and characterized by restriction endonuclease mapping. Southern analysis of genomic digests prepared with different restriction enzymes confirmed the same pattern for the chromosomal sequences. Transformants with the isolated plasmids had a phosphoglucose isomerase activity increased by a factor of 7. The cloned sequence hybridized to a constitutively synthesized 2.2 kb RNA in Northern analysis. The coding region includes a 2.05 kb EcoRI fragment common to all four inserts. A fragment including part of the PGI1 region was subcloned into vector YRp7 and used to induce integration at the PGI1 locus. Genetical and Southern analysis of stable transformants showed that single as well as tandem integration took place at this locus. This showed that the PGI1 gene had been isolated. Finally, and in contrast to the results of Kempe et al. (1974a, b) who reported three isoenzymes in yeasts, only one copy of the PGI1 gene per genome was found in several laboratory strains tested by Southern analysis.     

RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3–1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.     

Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae

Summary The structural gene PG11 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae. Plasmids carrying the LEU2 gene between genomic regions flanking the PG11 gene were constructed and used to transform a PGI1/pgi1 diploid strain. Stable transformants lacking the PGI1 allele were isolated. Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PG11 coding region were viable. Thus, the PGI1 gene is not essential in yeasts. However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1 haploid strains when fructose was supplied as sole carbon source. The wild-type growth rate could be restored by adding 0.1% glucose to the medium. Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose. Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5%. Also the pgi1 strains did not grow in glucose as sole carbon source. On the other hand pgi1/pgi1 diploid strains did not sporulate on the usual acetate medium. This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium. Under these conditions the pgi1 mutants sporulated with an efficiency of 25% compared with the wild type. These results suggest that (a) the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, (b) glucose-6-P is essential in yeasts, and (c) the oxidation of glucose-6-P through the glucose-6-P dehydrogenase reaction is not sufficient to support growth in yeasts.     

Bifunctional phosphoglucose/phosphomannose isomerase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrobaculum aerophilum</Emphasis>

ORF PAE1610 from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was first annotated as the conjectural pgi gene coding for hypothetical phosphoglucose isomerase (PGI). However, we have recently identified this ORF as the putative pgi/pmi gene coding for hypothetical bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). To prove its coding function, ORF PAE1610 was overexpressed in Escherichia coli, and the recombinant enzyme was characterized. The 65-kDa homodimeric protein catalyzed the isomerization of both glucose-6-phosphate and mannose-6-phosphate to fructose-6-phosphate at similar catalytic rates, thus characterizing the enzyme as bifunctional PGI/PMI. The enzyme was extremely thermoactive; it had a temperature optimum for catalytic activity of about 100°C and a melting temperature for thermal unfolding above 100°C.     

Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.     

Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of the plant gene encoding cytosolic phosphoglucose isomerase

The gene encoding a cytosolic isozyme of phosphoglucose isomerase (PGI, EC 5.3.1.9) was isolated from Clarkia lewisii, a wild flower native to California, and the structure and sequence of the entire coding region determined. PGI catalyzes an essential step in glycolysis and carbohydrate biosynthesis in plants. Spanning about 6 kb, the gene has 23 exons and 22 introns, the highest number yet reported in plants. The exons range in size from 43 to 156 nt and encode a protein of 569 amino acids. The protein is about 44–46% identical to the inferred protein sequences of pig, Escherichia coli and Saccharomyces cerevisiae. All of the introns are bordered with the consensus 5-GT...AG-3 dinucleotides. The longest intron includes a large stem-loop structure bounded by a perfect 9 nt direct repeat. We cloned the PGI gene from a genomic library prepared from a single plant of known PGI genotype. The locus and allele of the clone were identified by matching restriction fragments to fragments from genetically defined genomic DNAs by Southern hybridization.     

Characterization of mutations that overcome the toxic effect of glucose on phosphoglucose isomerase less strains of Saccharomyces cerevisiae

Abstract Glucose inhibits growth of yeast phosphoglucose isomerase mutants in permissive media. Mutants insensitive to this effect were isolated by selection on media containing 2% fructose + 2% glucose. A nuclear, monogenic, recessive mutation named rgl was responsible for this phenotype. The mutants isolated belonged to two complementation groups and have been termed rgl1 and rgl2 . When the double mutants were grown on fructose, fermentation of fructose or glucose was similar to that of the parental pgi strain but was not measurable when grown on fructose + glucose. Under these conditions, respiration of glucose and to a lesser extent of fructose was enhanced. The double mutants pgi rgl did not grow on fructose + glucose in the presence of antimycin A or ethidium bromide and their cytochrome oxidase was no longer sensitive to glucose repression. The results are interpreted as an indication that in the double mutants the glucose may be channeled through the pentose phosphate pathway to respiration.     

Molecular cloning and characterization of the trpC gene from Penicillium chrysogenum

Summary We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene ecodes a 2.7 kb poly(A)⁺ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal protion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation.     

Isolation, Nucleotide Sequence, and Physiological Relevance of the Gene Encoding Triose Phosphate Isomerase from Kluyveromyces lactis

Lack of triose phosphate isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae ΔTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.