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.     

An unstable mutant gene of the am locus of Neurospora results from a small duplication

We have cloned the unstable am mutant gene, am126, as well as the am gene from an am126 revertant. The mutation is a result of a 33-bp duplication that repeats a sequence starting 13 bp upstream of the 3' splice junction between intron 1 and exon 2 and extends 20 bp into exon 2. In addition, there is a G----A transition 2 bp upstream of the first copy of the duplicated sequence. In the revertant gene the wild-type sequence is precisely recovered, involving both the loss of the duplication and a reversion (A----G) of the associated transition. Our data suggest that only the more 5' of the two 3' splice junctions present in the duplicated version of the gene is used. This favors a 5'----3' scanning mechanism for exon splicing.     

Transformation of Aspergillus nidulans using the argB gene

Transformation of Aspergillus nidulans has been achieved using a chimeric vector comprising Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans DNA. Protoplasts of argB^? strains (defective for the ornithine carbamoyl transferase [carbamoylphosphate: l-ornithine carbamoyltransferase, EC 2.1.3.3] gene) of A. nidulans were incubated with plasmid pSal43 containing the cloned argB⁺ gene in the presence of poly(ethylene glycol) and CaCl₂. Transformant progeny was of three types; the majority were small slow-growing colonies which were non-viable when transferred to MM. The remaining large colonies, which were recovered at a frequency of 50 μg^?1 DNA in the best experiments, made up the other two types. One group were mitotically stable, showing no evidence of instability; the other comprised unstable types which segregated apparent transformant and parental phenotypes. The apparent transformants showed similar segregational properties. Southern hybridizations with a stable transformant suggested that it arose following integration of the argB⁺ at the arg locus. Analysis of an unstable transformant suggested that possibly more than one copy of the plasmid was integrated and then subjected to rearrangement.     

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.     

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.     

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.     

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.     

Cloning and characterization of a plasmid DNA from anacystis nidulans 6301

A plasmid DNA of Anacystis nidulans 6301 was isolated by CsCl-EtBr centrifugation. The Mr of the plasmid, named pBA1, was estimated to be 5.04 +/- 0.26 X 10(6) by electron microscopic analysis and 5.2 X 10(6) by agarose gel electrophoresis. The pBA1 DNA was opened at a unique site with BamHI and cloned in pBR322 vector propagated in Escherichia coli HB101 cells. The recombinant plasmid, named pBAS18, was digested with various restriction endonucleases and its cleavage map was constructed. Based on this result, the cleavage map of the pBA1 plasmid is presented.     

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.     

Cloning of a gene required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli

A clone bank, consisting of approx. 8100 colonies, has been created for the spirochete Leptospira biflexa serovar patoc in Escherichia coli using pBR322 as the vector. One of these clones contains the genetic information needed to complement a defect in the trpE gene of E. coli. The information resides on a 20.5-kb plasmid designated pYCl, which carries a 16-kb insert consisting of three HindIII fragments. It does not complement defects in other genes needed for the biosynthesis of tryptophan in E. coli.     

Isolation and characterization of pock-forming plasmids for Streptomyces griseus from soil actinomycetes

Thirty independent actinomycetes strains carrying plasmids were isolated from soil. These plasmids were purified as cccDNA by CsCl-EtBr equilibrium density-gradient centrifugation. Plasmids that induce "pocks", namely formation of circular zones of sporulation-inhibition, were selected by protoplast transformation of streptomycin-producing strain, Streptomyces griseus ATCC10137. Six pock-forming plasmids, pOA7, pOA11, pOA15, pOA23, pOA29 and pOA30, were obtained, and their cleavage maps, transformation frequencies, and copy numbers, as well as their stability, are described.     

Isolation of the origin of replication of the IncW-group plasmid pSa

The origin of replication of the IncW plasmid pSa has been cloned and the function of this origin in Escherichia coli examined. A 1.9-kb region of DNA is required for efficient autonomous replication, and a 0.47-kb fragment within this region can initiate replication only in the presence of an autonomously replicating derivative of pSa. An Mr 35,000 protein (repA) is encoded adjacent to the origin and is required for efficient initiation of replication. The derivatives examined provide information suggesting a direct role of partition factors in plasmid replication and incompatibility.     

The making of strand-specific M13 probes

A novel approach has been developed for the preparation of highly radioactive, strand-specific M13 probes. A universal primer, complementary to the region 5' to the multiple cloning sites of M13mp7, was used to initiate the DNA synthesis of the complementary strand of the M13 sequence downstream from the inserted sequence. The synthesis of the (?) strand, which was labeled with a radioactively labeled precursor, did not proceed to completion so that the inserted sequence was kept single-stranded. Thus, a partially double-stranded probe that had the specificity of this inserted sequence was obtained. As an example for the application of single-stranded specific hybridization probes, an M13mp7 subclone of a zein cDNA clone of maize (A30) was labeled and used in a dot hybridization test to select from the hundreds of M13mp7 subclones of the zein genomic clone, 24, the sequences complementary to the probe. The specificity of the probe was confirmed by dideoxy chain terminator sequencing experiments.     

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 of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production

The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments. They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment. The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9. A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers. The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.     

Mitochondrial DNA from lupine: restriction analysis and cloning of fragments coding for tRNA

Mitochondrial DNA (mtDNA) was isolated from lupine. Restriction analysis was used to estimate its size, which is about 180 kb. A BamHI bank of this mtDNA was constructed using plasmids pBR322 and pBR327 as vectors. Eight clones containing plasmids hybridizing to mitochondrial tRNA (mttRNA) were isolated. Restriction maps of these plasmids were determined. Six of these plasmids hybridized to unique fragments and two to two fragments of very similar size, all obtained by BamHI cleavage of mtDNA.     

Comparison of sequence repetitiveness of human cDNA and genomic DNA using the miniplasmid vector, piVX

We studied the sequence repetitiveness of human cDNA and genomic DNA fragments inserted in the miniplasmid piVX. Sequence repetitiveness was assayed by the frequency with which a given insert mediated recombination between the chimeric miniplasmid and a recombinant bacteriophage library constructed from large random human genomic fragments. The methodology allows rapid analysis and isolation of sequences of a given copy number in the genome: few (1 to 10 copies), low order-repeated (10 to 100 copies) and a more highly repeated (over 100 copies). In a model application of the method, the distribution of these classes of sequences was compared in cDNA and genomic DNA libraries constructed in piVX. The major difference observed between cDNA and genomic DNA repeat structure was the paucity of highly repeated elements in cDNA copies from high-molecular-weight cytoplasmic poly(A) + RNA.