Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium.     

Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.     

Sequence analysis of a Dictyostelium discoideum gene coding for an active dihydroorotate dehydrogenase in yeast

A Dictyostelium discoideum DNA fragment isolated on the basis of its ability to complement the ural mutation of yeast, codes for a dihydroorotate dehydrogenase activity. The complete nucleotide sequence of this 1898 bp fragment has been determined and reveals an open reading frame capable of coding for a 369 amino acid polypeptide of molecular mass 47.000. The gene shows preferential use of codons with weak pairing forces. Eleven codons, mainly those with a G in the third position, are absent. The flanking sequences are unusually rich in A + T (80%). Several direct and inverted repeats exist in the 5' flanking sequence.     

Molecular cloning and sequencing of the beta-isopropylmalate dehydrogenase gene from the cyanobacterium Spirulina platensis.

The gene for beta-isopropylmalate dehydrogenase (EC 1.1.1.85) of Spirulina platensis (leuB) was cloned from a lambda EMBL3 genomic library by heterologous hybridization using the Nostoc UCD 7801 leuB gene as a probe. The sequence of the entire leuB coding region was determined as well as 645 bp of 5' flanking region and 956 bp of 3' flanking region. DNA sequencing revealed an open reading frame of 1065 nucleotides capable of encoding a polypeptide of 355 amino acids. Homologies between the amino acid sequence deduced from the nucleotide sequence of the S. platensis leuB gene and the amino acid sequences published for corresponding proteins either from bacteria or yeasts are 45% or more. Northern hybridization analysis indicated that the S. platensis leuB gene is transcribed as a single monocistronic RNA of approximately 1200 bases.     

A 3'' co-terminal family of mRNAs from the herpes simplex virus type 1 short region: two overlapping reading frames encode unrelated polypeptide one of which has highly reiterated amino acid sequence.

We have used DNA sequencing, mRNA mapping and in vitro translation to characterise three partially overlapping genes in the genome of herpes simplex virus (HSV) type 1. These genes specify three mRNAs with distinct 5' termini but a common 3' terminus, the longest of which is immediate-early (IE) mRNA-5. The 12,000 MW (12K) IE polypeptide encoded by IEmRNA-5 is translated from an 88 codon open reading frame, leaving a 1200 base 3' non-translated region. The second mRNA (mRNA-B) is initiated within the coding sequence of IEmRNA-5, and encodes a 21K polypeptide. The 12K and 21K polypeptide coding regions do not overlap. The third mRNA (mRNA-C) is initiated within the coding region of mRNA-B, and encodes a 33K polypeptide. The reading frame for 33K has a 110 codon out-of-frame overlap with the 21K reading frame. This is the first instance of overlapping genes described for HSV. The 21K polypeptide is thought to be a DNA binding protein and is remarkable for an array of 24 tandem repeats of the sequence X/Pro/Arg (where X represents predominantly Glu, Asp, Thr, Ser or Val) in its C-terminal portion. This array, which occupies most of the region of overlap with 33K, can vary in repeat number between virus strains.     

Sequence and deletion analysis of the recombination enhancement gene (ref) of bacteriophage P1: evidence for promoter-operator and attenuator-antiterminator control.

The ref gene of bacteriophage P1 stimulates recombination between two defective lacZ genes in the Escherichia coli chromosome (lac x lac recombination) and certain other RecA-dependent recombination processes. We determined the DNA sequence of the 5' portion of the ref gene and tested various regions for functionality by inserting DNA fragments lacking increasing amounts of 5' sequence into plasmid and lambda phage vectors and measuring the ability of the constructs to stimulate lac x lac recombination. The region found essential for Ref activity in the absence of external heterologous promoters encodes two presumptive promoters, pref-1 and pref-2, whose -10 regions fall in a nearly perfect 13-base-pair (bp) tandem repeat. The -10 region of the putative pref-1 is part of a phage P1 c1 repressor recognition sequence. The first two ATG codons in the ref reading frame are, respectively, 90 and 216 bp downstream from the putative promoter-operator region. Deletion analysis indicated that translation can initiate at either ATG (although neither is associated with a canonical ribosome-binding sequence) and that the 42 amino acids in between are not indispensable for Ref stimulation of lac x lac recombination. However, the shorter reading frame appears to encode a less active polypeptide. The 91-bp leader region between the putative promoter-operator and the first ATG contains 30 codons in frame with the ref structural sequence, but its frame can be shifted without affecting Ref activity. The leader region ends with an apparent rho-independent termination sequence (attenuator). Deletion of 18 bp of early leader sequence drastically reduced Ref activity, even when ref was driven by a heterologous promoter (plac). An 8-bp internal deletion in the putative attenuator sequence relieved this requirement for the early leader sequence. This latter observation, along with nucleotide complementarity between portions of the early leader and attenuator sequences, are consistent with preemption of attenuation by the early leader.     

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.