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.     

The nucleotide sequence of the yeast ARG4 gene

The complete nucleotide sequence of a 2296-bp DNA fragment containing the yeast (Saccharomyces cerevisiae) ARG4 gene has been determined. This gene specifies the synthesis of the arginine biosynthetic enzyme, argininosuccinate lyase (EC 4.3.2.1). The sequence contains one major open reading frame of 463 codons, giving a calculated M_r of 52010 for the protein, in good agreement with the experimentally determined value of 53 000. The sequence upstream from the ARG4 gene shares structural features in common with other yeast genes subject to general amino acid control.     

Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.     

Comparative study of the L1 family in the genus Mus. Possible role of retroposition and conversion events in its concerted evolution

The long interspersed repetitive family L1 was analysed in different species belonging to the genus Mus. It is shown to be highly conserved even in M.n. setulosus, which diverged from the other species around ten million years ago. The study of the linkage between diagnostic restriction sites in the various species and the sequence variations of different regions of the L1Md repeat shows that the L1 family undergoes concerted changes involving subsets of repeats. The rate at which this homogenization process occurs does not appear to be the same for all the subfamilies detected. The L1Md repeat in the twelfth intron of the serum albumin gene of Balb/c mice is shown to be a recent insertion. The role retroposon- and gene conversion-like events may play in the concerted evolution of the L1 family is discussed.     

Cloning of the vaccinia virus telomere in a yeast plasmid vector

The vaccinia virus DNA telomere, which contains a covalently closed hairpin structure, has been cloned in a yeast plasmid vector. Restriction mapping indicates that the cloned vaccinia telomere is maintained in yeast not in its native hairpin configuration but as an inverted repeat structure, within a circular plasmid, with the sequences of the viral hairpin now at the axis of symmetry of an imperfect palindrome. As such, the cloned telomere resembles the telomeric replicative intermediate observed during vaccinia virus DNA replication. Small deletions and duplications in the viral inverted repeats of different clones suggest a model in which the observed circular plasmids were generated in yeast by the replication of hybrid linear DNA molecules consisting of the linearized yeast vector flanked by two hairpin-containing vaccinia termini.     

The ori sequences of the mitochondrial genome of a wild-type yeast strain: number,location, orientation and structure

We have investigated the number, the location, the orientation and the structure of the seven ori sequences present in the mitochondrial genome of a wild-type strain, A, of Saccharomyces cerevisiae. These homologous sequences are formed by three G + C-rich clusters. A, B and C, and by four A + T-rich stretches. Two of the latter, p and s, are located between clusters A and B; one, l between clusters B and C; and one r, either immediately follows cluster C (in ori 3–7), or is separated from it by an additional A + T-rich stretch, r', (in ori 1 and ori 2). The most remarkable differences among ori sequences concern the presence of two additional G + C-rich clusters,β and γ, which are inserted in sequence l of ori 4 and 6 and in the middle of sequence r of ori 4,6 and 7, respectively. Neglecting clusters /gb and γ and stretch r', the length of on sequences is 280 ± 1 bp, and that of the l stretch 200 ± 1 bp. Hairpin structures can be formed by the whole A-B region, by clusters β and γ, and (in ori 2–6) by a short AT sequence, lp, immediately preceding cluster /gb. An overall tertiary folding of ori sequences can be obtained. Some structural features of ori sequences are shared by the origins of replication of the heavy strands of the mitochondrial genomes of mammalian cells.     

The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli

The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.     

Cloning and expression of Clostridium pasteurianum galactokinase gene in Escherichia coli K-12 and nucleotide sequence analysis of a region affecting the amount of the enzyme

The Clostridium pasteurianum galactokinase gene was cloned by complementation, of the galK locus, into Escherichia coli. Restriction enzyme analysis subcloning and Tn5 mutagenesis indicated that the gene was located on a 1.8 X 10(3) base-pair ClaI-Sau3A fragment that encoded a polypeptide of approximately 40 Mr. Although the C. pasteurianum and the E. coli galactokinases have similar subunit molecular weights, Southern hybridization analysis indicated no strong homology between their genes. Even though this clone showed a low level of galactokinase expression, the Gal+ phenotype, provided by the clostridial galactokinase, was unstable in E. coli, and the gene was frequently inactivated by the spontaneous acquisition of insertion sequences. A second clone containing this gene on a large restriction fragment was isolated by hybridization. This clone was unable to grow on galactose-containing media due to the overproduction of galactokinase. Comparison of the plasmids from these two clones revealed that the second contained an additional 300 base-pairs located at one end of the galactokinase gene. Appropriate operon fusions with a promoter-less E. coli galactokinase gene indicated that these additional 300 base-pairs had promoter activity in E. coli. The DNA sequence of this region which lies upstream of the C. pasteurianum galactokinase gene was determined and compared with that from several clones producing high, low or undetectable amounts of galactokinase. The reasons for the high and low level expression and for the instability of the C. pasteurianum galactokinase in E. coli are discussed. The presence of the galactokinase suggests that galactose is used in C. pasteurianum through the Leloir pathway via galactose 1-phosphate.     

Tn5 mutagenesis of the transforming genes of human adenovirus type 5

We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn 5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCDl, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region la and the N-terminal region of early region 1b are essential for DNA-mediated transformation.     

Cloning and expression of an extracellular-agarase gene from Streptomyces coelicolor A3(2) in Streptomyces lividans 66

An extracellular agarase gene was cloned from Streptomyces coelicolor A3(2) strain M130 into S. lividans 66 using the multicopy plasmid vector pIJ702. Various deletion derivatives of the initial clone (pMT605) were obtained by in vitro and in vivo methods. This allowed the gene to be localised to a 1.9-kb segment of DNA. The agarase enzyme was overproduced (up to 500 times) and exported efficiently into the medium. The agarase protein was identified as a 28-kDal band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE); in the case of one derivative, pMT608, this band accounted for nearly 50% of the total extracellular protein. Differences in agarase production between the deletion derivatives correlated well with plasmid stability.     

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3′-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated M_r of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).     

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.     

Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322

The Tn10 tetracycline resistance gene, tetA, encodes a tetracycline-inducible protein with an apparent Mr of 36 X 10(3). We have determined the nucleotide sequence of the Tn10 tetA gene. The extent of the tetA gene was determined by analysis of amino-terminal and carboxy-terminal deletion mutants. We conclude that a single Tn10 gene, the tetA gene, is sufficient to confer tetracycline resistance. The predicted Mr of the tetA protein is 43.2 X 10(3). The sequence homology between the Tn10 tetA gene and the pBR322 tetracycline resistance determinant (49% nucleotide homology, 44% amino acid homology) indicates that these phenotypically distinct tetracycline-resistance determinants must have evolved from a common ancestral sequence. The markedly hydrophobic character of the predicted amino acid sequences of the Tn10 tetA and pBR322 tet-coded proteins suggests that a substantial portion of these proteins may be embedded within the cytoplasmic membrane.     

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.     

Genetic and physical analysis of the thioredoxin (trxA) gene of Escherichia coli K-12

The trxA gene of Escherichia coli K-12 has been cloned into multicopy plasmids on DNA fragments of varying sizes. The smallest of these was a 1-kb fragment resulting from partial digestion with Sau3A (pBHK10). The complete nucleotide sequence of the trxA gene and its promoter was determined. Comparison of the DNA sequence with the published amino acid sequence revealed the inversion of two amino acid pairs and the possibility of a leader peptide 18 amino acids in length. Three-factor P1 transductional crosses and physical mapping experiments have determined a map order of ilv-trxA-uvrD-corA-metE.     

Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.