A gene encoding a DnaK/hsp70 homolog in Escherichia coli.

Eukaryotic organisms have been shown to have multiple forms of hsp70-class stress-related proteins, but only a single family member, DnaK, has been found in prokaryotes. We report here the identification of a heat shock cognate gene, designated hsc, in Escherichia coli. The amino acid sequence deduced from hsc predicts a 65,647-Da polypeptide having 41% sequence identity with DnaK from E. coli, and overexpression produces a protein (Hsc66) with properties similar to DnaK. In contrast to dnaK, however, the hsc gene lacks a consensus heat shock promoter sequence, and expression is not induced by elevated temperature. The hsc gene is located near 54 min on the physical map, immediately upstream of the fdx gene, which encodes a [2Fe-2S] ferredoxin; evidence is presented that the hsc and fdx genes make up a bicistronic operon in which expression of the ferredoxin is coupled to that of Hsc66. The function of Hsc66 is not known, but the coregulation of its expression with that of ferredoxin suggests the possibility of a specific role in association with the ferredoxin protein.

Isolation and characterization of the Schizosaccharomyces pombe rad3 gene, involved in the DNA damage and DNA synthesis checkpoints.

We have cloned the Schizosaccharomyces pombe rad3 gene which is involved in G2 arrest following DNA damage, and in the dependence of mitosis on the completion of DNA replication. The gene was cloned by complementation of the sensitivity to UV light and gamma rays of the rad3-136 mutant with an Sz. pombe genomic library. Sublocalization of the complementing activity and sequencing of the clone identified an intronless 3210-bp open reading frame capable of encoding a 1070-amino acid protein with an M(r) of 121974. The rad3 gene is a new gene with no homologs in existing sequence databases. The gene is poorly expressed, with a codon bias index of -0.01. A disruption mutant affecting the coding region was only slightly more sensitive to UV light than the original rad3-136 mutant. The rad3 gene was mapped to NotI fragment C on chromosome II.

Expression of human ferredoxin in Saccharomyces cerevisiae: mitochondrial import of the protein and assembly of the [2Fe-2S] center.

Vertebrate ferredoxins function in the transfer of reducing equivalents from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes involved in steroid metabolism. We report here the expression of human mitochondrial ferredoxin in the yeast Saccharomyces cerevisiae. The full-length ferredoxin protein containing the ferredoxin mitochondrial leader sequence could not be stably expressed in S. cerevisiae, but a fusion protein consisting of the mature portion of ferredoxin linked to the mitochondrial leader sequence of the S. cerevisiae cytochrome c oxidase subunit Va protein (COX5a) could be stably expressed. The COX5a:ferredoxin fusion protein was targeted to the mitochondria as a preprotein and was cleaved at the normal processing site of the COX5a presequence during import into the matrix. Absorption spectra and electron transfer activity of the isolated fusion protein established that the [2Fe-2S] center was correctly assembled and incorporated into the recombinant ferredoxin in this heterologous system.

Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe.

We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination. The coding region of the gene is 582 base pairs long and contains no introns. The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues. This gene does not exhibit significant homology to any other known repair gene. The major transcription start site is at 27 base pairs upstream of the putative start codon. Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity. A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant. Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence. This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product. We have localized the rad1 gene to NotI fragment E of the S. pombe genome.