The nucleotide sequence of the promoter region of hisS, the structural gene for histidyl-tRNA synthetase

A plasmid has been constructed which carries hisS, the structural gene for histidyl-RNA synthetase of E. coli, on a 1.6-kb fragment bounded by PvuII and BstEII sites. The DNA sequence of both ends of this fragment was determined. The amino-terminal sequence of histidyl-tRNA synthetase was also determined to locate the promoter proximal coding region and the frame in which it is read. Three promoters were identified by consensus criteria. The region surrounding these promoters contains extensive twofold symmetry.     

The complete nucleotide sequence of a 16S ribosomal RNA gene from tobacco chloroplasts: Recombinant plasmid; sequence homology; stem and loop structure

The complete nucleotide sequence of a 16S ribosomal RNA gene from tobacco chloroplasts has been determined. This nucleotide sequence has 96% homology with that of maize chloroplast 16S rRNA gene and 74% homology with that of Escherichia coli16S gene.The 3′ terminal region of this gene contains the sequence ACCTCC which is complementary to sequences found at the 5′ termini of prokaryotic mRNAs.The large stem and loop structure can be constructed from the sequences surrounding the 5′ and 3′ ends of the 16S gene. These observations demonstrate the prokaryotic nature of chloroplast 16S rRNA.     

Lethality of palindromic DNA and its use in selection of recombinant plasmids

A plasmid derived from ColE1 is constructed so that the removal of one restriction endonuclease HindIII fragment allows the ends of the remaining single fragment (the replicator) to be joined, generating a palindromic sequence 2394 bp in length. The circular species thus produced gives rise to transformants of E. coli at very low frequency. Since the palindromic sequence is effectively lethal to a plasmid containing it, the replicator will give rise to more transformants when the restriction fragment originally removed from it is replaced by another. This principle can be exploited to allow the efficient molecular cloning of unselected restriction fragments.     

The nucleotide sequence of IS5 from Escherichia coli

A 3-kb fragment of Haemophilus haemolyticus DNA which carries the HhaII restriction (r) and modification (m) genes has been cloned into the PstI site of pBR322 (Mann et al., 1978). When propagated in Escherichia coli, it was observed that spontaneous insertions of IS5 inactivated the restriction gene, producing r- mutants at a frequency of 10(-6). Electron microscopy, restriction-site mapping and sequence analysis of two r- plasmids have demonstrated the presence of IS5 at a single target site in both possible orientations. The complete nucleotide sequence of IS5 has been determined. It is 1195 bp long and has inverted terminal repeats of 16 bp. The target site for IS5 in this plasmid is 5'-CTAG. Approx. ten copies of IS5 were found to be present at about the same locations on the E. coli chromosome in various K-12 strains, using Southern hybridization analysis.     

Cloning of randomly sheared DNA fragments from a phi 105 lysogen of Bacillus subtilis identification of prophage-containing clones

A gene bank for Bacillus subtilis has been developed by cloning randomly sheared DNA fragments of a B. subtilis (phi 105) lysogen DNA in Escherichia coli employing the pMB9 plasmid vector. The DNA was inserted by the oligo(dA)-oligo(dT) method, and the average insert size of the cloned DNA was 7 kilobase pairs (kb). Three clones have been identified which carry DNA from the phi 105 prophage. None of these clones contain the phage-chromosome junction.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.     

Heteroduplex analysis of tRNA3bVal genes from the 90BC and 84D sites of Drosophila melanogaster

Two recombinant DNA plasmids coding for Drosophila tRNA_3b^Val hybridize to two different loci on the Drosophila genome. Plasmid pDt41R hybridizes at the 90BC site, and pDt78R hybridizes at the 84D site. Heteroduplexes of the two plasmids were examined by electron microscopy. The study shows that the Drosophila segments have significant homology only over the length of the tRNA_3b^Val genes.     

Cloning and characterization of a genomic DNA fragment carrying the basic copy of the gene coding for variant surface antigen 118 of Trypanosoma brucei

It has been proposed (Hoeijmakers et al., 1980b) that variant surface antigen (VSA) gene expression in Trypanosoma brucei is accomplished by a gene re-arrangement involving the basic copy of the VSA gene to give the so-called expression-linked copy (which is present only in the strain expressing that particular antigen). In this publication, the basic and expression-linked copies of the gene have been visualized by Southern blot analysis of nuclear DNA and shown to be located on HindIII fragments of 4.5 and 10-12 kb, respectively. In addition, several other bands of weaker hybridization are seen, probably representing evolutionary relatives. Using a shotgun approach, HindIII gene banks have been constructed and recombinants isolated which carry the 4.5-kb HindIII fragment containing the VSA118 gene basic copy. Several clones containing evolutionary relatives were also found. The 4.5-kb HindIII fragment is able to hybridize to probes derived from both the 5' and 3' ends of the cDNA, while the relatives have homology only to the 3' end. A detailed comparison of the restriction map of VSA118 cDNA with that of the VSA118 basic copy showed no differences, demonstrating that the gene contains no introns. This result also indicates that the gene from which VSA118 mRNA is transcribed (whether this be the basic copy or the expression-linked copy) is identical to the basic copy over the region analysed.     

The ntr genes of Escherichia coli activate the hut and nif operons of Klebsiella pneumoniae

Regulation of the synthesis of glutamine synthetase and of the arginine and glutamine transport systems (Ntr phenotype) in Salmonella have been shown to require two regulatory genes on the C-terminal side of the glnA gene (McFarland et al., 1981). We have cloned a HindIII-EcoRI DNA fragment from Escherichia coli coding for analogous properties with respect to the Ntr phenotype in E. coli. A plasmid containing this E. coli DNA fragment joined to another fragment carrying a cyanobacterial glnA gene (but no functional regulatory genes) was introduced into a Klebsiella pneumoniae mutant with a Gln-Ntr- phenotype, i.e., which could not derepress nitrogenase. The cyanobacterial gene made the Klebsiella strain Gln+ and the E. coli DNA fragment made the strain Ntr+, including the ability to derepress nitrogenase fully. Thus the products of the glnA-linked ntr genes of E. coli can regulate expression of the Ntr-dependent genes of Klebsiella.     

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.     

Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12

The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb. This plasmid does not fully complement a uvrA mutation in a Rec+ background. Plasmids which contain the uvrA and ssg genes, but not the lexA gene, show a higher but still only partial complementation. Full complementation achieved when the ssb gene us inactivated by insertion of Tn5. Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light. The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed.     

Isolation and characterization of rat ribosomal DNA clones

Four EcoRI fragments, which contain the transcribed portion of the rat rDNA repeat, have been isolated from a rat genome library cloned in lambda Charon 4A vector. Three of the fragments, 9.6, 6.7, and 4.5 kb, from clones lambda ChR-B4, lambda Nr-42, and lambda ChR-C4B9, contained part of the 5'-NTS, the 5'-ETS, 18S rDNA, ITS-1, 5.8S rDNA, 28S rDNA and approximately 3.5 kb of the 3'-NTS. Two EcoRI fragments, from clones lambda ChR-B4 and lambda ChR-B7E12, which coded for the 5'-NTS, the ETS, and most of the 18S rDNA, differed by 1 kb near the EcoRI site upstream of the 5' terminus of 18S rRNA. Restriction maps of the cloned DNA fragments were constructed by cleavage of the fragments with various restriction endonucleases and Southern hybridization with 18S, 5.8S, and 28S rRNA. These maps were confirmed and extended by subcloning several regions of the repeat in pBR322.