Nucleotide sequence of plasmid NAH7 gene nahR and DNA binding of the nahR product.

The nah and sal operons of the 80-kilobase-pair (kb) NAH7 plasmid specify catabolism of naphthalene and salicylate under positive regulation by gene nahR. A 1.75-kb fragment (PstI-HindIII) cloned into the pCP13 derivative of vector RK2 complemented in trans five nahR mutations. The fragment sequence contained a 1,122-base-pair open reading frame with a predicted sequence of 374 residues that was rich in basic amino acids with regions similar to known DNA-binding proteins. Clones from the nahR gene region were expressed in mexicells. Plasmid pY1923, carrying the 1.75-kb PstI-HindIII fragment, expressed a protein of Mr ca. 35,000 which bound to the upstream region of gene nahR in a gel electrophoresis DNA-binding assay. Other clones expressed proteins of currently unknown function; pY1311, with the 1.1-kb HindIII fragment, produced a polypeptide with an Mr of 23,000, and pY1812, with the 1.2-kb PstI-SphI fragment, produced a polypeptide (Mr 41,000) which appeared to be a fused nahR-lacZ product.     

Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product

The dnaJ and dnaK genes are essential for replication of Escherichia coli DNA, and they constitute an operon, dnaJ being downstream from dnaK. The amount of the dnaJ protein in E. coli is substantially less than that of the dnaK protein, which is produced abundantly. In order to construct a system that over-produces the dnaJ protein, we started our study by determining the DNA sequence of the entire dnaJ gene, and an operon fusion was constructed by inserting the gene downstream of the lambda PL promoter of an expression vector plasmid, pPL-lambda. Cells containing the recombinant plasmid produced dnaJ protein amounting to 2% of the total cellular protein when cells were induced. The overproduced protein was purified, and Edman degradation of the protein indicated that the NH2-terminal methionine was found to be processed. From the DNA sequence of the dnaJ gene, the processed gene product is composed of 375 amino acid residues, and its molecular weight is calculated to be 40,975.     

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.     

Cloning, structure, and expression of the Escherichia coli K-12 hisC gene.

We used an expression vector plasmid containing the Escherichia coli K-12 histidine operon regulatory region to subclone the E. coli hisC gene. Analysis of plasmid-coded proteins showed that hisC was expressed in minicells. A protein with an apparent molecular weight of 38,500 was identified as the primary product of the hisC gene. Expression was under control of the hisGp promoter and resulted in very efficient synthesis (over 100-fold above the wild-type levels) of imidazolylacetolphosphate:L-glutamate aminotransferase, the hisC gene product. The complete nucleotide sequence of the hisC gene has been determined. The gene is 1,071 nucleotides long and codes for a protein of 356 amino acids with only one histidine residue.