Daniel Vapnek

Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenyltransferase

Abstract The nucleotide sequence of 1400 bp from R-plasmid R538-1 containing the streptomycin/ spectinomycin adenyltransferase gene ( aadA ) was determined, and the location of the aadA gene was identified by a combination of insertion and deletion mutants. Its gene product, aminoglycoside 3″-adenyltransferase (AAD(3″)(9), has a M r of 31,600.

Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Abstract Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases Bam HI or Eco RI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases Bam HI, Eco RI, Hin dIII and Hpa I. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a Bam HI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.

Electron microscopic analysis of bacteriophages P1, P1Cm, and P7. Determination of genome sizes, sequence homology, and location of antibiotic-resistance determinants.

Abstract The sizes of the genomes of bacteriophages P1, P1Cm, and P7 (φAMP) and their corresponding plasmid prophages were measured by electron microscopy. It was found that the genomes from the phage particles were the same size, while the prophage DNA molecules of P1Cm and P7 were larger than the P1 prophage by an amount close to the size of the antibiotic determinant which they carried. Heteroduplex analysis of hybrids formed in vitro between P1 and P1Cm DNA showed one region of nonhomology, an insertion of 2.2 kilobases (kb) which was presumed to be the Cm r determinant. Similar hybrids between P1 and P7 DNA showed six regions of nonhomology, two insertion-deletion loops, and four substitution loops. Further analysis showed that the insertion of a 5.5-kb segment contained the determinant for ampicillin resistance. Sequence homology between P1 and P7, determined from heteroduplex analysis, was about 90% with respect to the genome size of P1.

Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli

Abstract The prophages of the related temperate bacteriophages P1 and P7, which normally exist as plasmids, suppress Escherichia coli dnaA (ts) mutants by integrating into the host chromosome. The locations of the sites on the prophage used for integrative recombination were identified by restriction nuclease analysis and DNA-DNA hybridization techniques. The integration of P1 and P7 often involves a specific site on the host DNA and a specific site on the phage DNA; the latter is probably the end of the phage genetic map. When this site is utilized, the host Rec + function is not required. In Rec + strains, P1 and P7 may also recombine with homologous regions on the host chromosome; at least one of these regions is an IS1 element. In some integration events, prophage deletions are observed which are often associated with inverted repeat structures on the phage DNA. Thus, P1 and P7 may employ one of several different mechanisms for integration.

Versatile cloning vectors derived from the runaway-replication plasmid pKN402

Two cloning vectors have been constructed employing runaway-replication mutants of plasmid R1. One of these, pMOB45, carries tetracycline and chloramphenicol resistance. The other, pMOB48, carries chloramphenicol resistance, lacOP, and an assayable part of the lacPOZ operon. Both of these plasmids can be amplified to high levels by heat induction, which condition does not lead to inhibition of protein synthesis; thus the plasmid can produce large amounts of DNA and protein. In pMOB48, a unique BamHI site is present near the amino-terminus of the beta-galactosidase gene. Chimeras formed by the insertion of restriction fragments at this site can be detected on X-gal plates, and can be used for the lacIq-controlled expression of proteins which are fused to the amino-terminus of beta-galactosidase. Induction with IPTG at 40 degrees C leads to the synthesis of extremely high levels of proteins whose gene have been cloned into this site.

Isolation and characterization of cloned fragments of bacteriophage P1 DNA

Abstract Fragments of PI DNA generated by endo·R·EcoRI and endo·R·BamHI were mixed with appropriate cloning vectors (ColElApr, pBR313, pBR322) ligated in vitro and used to transform a nonsuppressing strain of Escherichia coli. In this way clones of 9 of the 26 EcoRI fragments and 5 of the 14 BamHI fragments were obtained. Marker rescue tests were used to ascertain which regions of the P1 genome were contained on the various DNA fragments. These cloned fragments are useful as probes for specific regions of the P1 genome.

Transcription and translation in E. coli of hybrid plasmids containing the catabolic dehydroquinase gene from Neurospora crassa

Two hybrid plasmids which carry the gene for Neurospora crassa catabolic dehydroquinase (C-DHQase) and complement an aroD6 (dehydroquinase-deficient) auxotroph of Escherichia coli have been analyzed. One of these contains a 2.9 kilobase (kb) fragment cloned in the HindIII site of plasmid pBR322 (pVK57) and the other contains a 6.8 kb fragment cloned in the PstI site (pVK88). Restriction enzyme mapping of these plasmids has demonstrated that the 2.9 kb fragment is totally contained within the 6.8 kb fragment. When the polarity of either the HindIII fragment or PstI fragment was reversed with respect to pBR322 no effect was observed on either the ability of the hybrid to complement an aroD- auxotroph or on the level of C-DHQase activity. In vivo transcription of plasmid pVK88 in both orientations was analyzed by RNA-DNA hybridization and by the techniques developed by Southern (1975). Approx. 40% of the plasmid-directed transcription occurred from the cloned PstI fragment and 60--70% of these N. crassa transcripts were encoded by the 2.9 kb HindIII fragment. The Southern technique allowed a further localization of the region of most extensive transcription to a 1.8 kb HindIII-EcoRI fragment. Biochemical analysis revealed that the C-DHQase protein produced by strains harboring pVK57 and pVK88 in either orientation was identical to the N. crassa enzyme. Furthermore, when these plasmids were segregated into minicells and labeled with 14C amino acids, the C-DHQase protein was synthesized at a level comparable to other plasmid-encoded proteins. Taken together, these experiments demonstrate that transcription is efficiently initiated in E. coli from a site on the cloned N. crassa DNA and that the resulting C-DHQase mRNA is efficiently and accurately translated.

Molecular cloning of restriction fragments and construction of a physical and genetic map of the Escherichia coli plasmid R538-1.

Abstract A genetic and physical map of Escherichia coli plasmid R538-1 was constructed using restriction endonucleases and molecular cloning techniques. R538-1 DNA was cleaved into 12 fragments by endonuclease · R · Eco RI, 6 fragments by endonuclease R · Hin dIII, and 3 fragments by endonuclease R · Bam HI. The order of these fragments was determined by standard restriction fragment mapping techniques. Endo · R · Eco RI, endo · R · Hin dIII, endo · R · Bam HI, and endo · R · Pst I fragments obtained from R538-1 and ColE1-derived plasmids (pMB9, ColE1Ap r , and pBR322) were ligated in vitro and used to transform E. coli C600. Transformants were selected for antibiotic resistance markers carried by R538-1. Analysis of the R538-1 fragments contained in these hybrid plasmids permitted the construction of a genetic map of the R538-1 plasmid. The genetic map of this plasmid is very similar to that of plasmid R100.

Transcription and translation of R-plasmid 538-1 DNA: Effects of mercury induction and analysis of polypeptides coded for by the r-determinant region

Abstract In vivo transcription and translation of R-plasmid 538-1 in Escherichia coli was analyzed. Transcription of individual restriction fragments was determined qualitatively by utilizing the techniques developed by Southern J. Mol. Biol. 98, 503–517, 1975 and quantitatively by carrying out DNA-RNA filter hybridization. The most active region of R-plasmid transcription in strains repressed for conjugal transfer was found to occur in the region of the R-plasmid carrying the antibiotic resistance genes. In plasmids derepressed for conjugal transfer, a high level of transcription from the transfer gene region was also observed. When strains carrying R538-1 were induced with Hg2+ a high level of transcription was observed from the region of the R-plasmid carrying the genes for Hgr. Hybrid ColE1 plasmids carrying restriction fragments from the antibiotic resistance region of R538-1 were segregated into minicells. Labeling of the minicells with [35S]methionine or [14C]amino acids allowed identification of the proteins coded by the fragments. A limited number of proteins were detected, and several of these have been correlated with the antibiotic resistance genes carried by R538-1.

Map location of the kanamycin resistance determinant in P1Km0

Abstract The kanamycin resistance determinant in P1Km lies between am 33 and gene 2 on the P1 genetic map. This is within the invertible region (C loop) of P1 DNA.