1887

Abstract

The structural genes and encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from subsp. (L. ). The genes were isolated after screening genomic sublibraries with specific and probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order . The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between and , and by only 18 bp between and . The codon usage in and three other glycolytic genes from differed noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gene, and a less abundant mRNA of 3.4 kb corresponding to the cluster. The absence of a visible terminator in the 3′-end of the shorter transcript and the location of this 3′-end inside the gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gene.

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1998-04-01
2021-04-23
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