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Biomedical subjects

B Sain

Publications and source records attributed to B Sain.

14 recordsLinked to original sources
PubMed1984

The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434.

A new plasmid vector, pNS1, is described that allows positive selection for bacterial transformants carrying recombinant plasmids. It is a derivative of pBR327, and it includes a regulatory region from the lambdoid phage 434. The expression of the TcR gene of pNS1 is under the control of the ORpR operator-promoter of phage 434, which is regulated by the repressor gene cI. The cloning sites of pNS1 (StuI, NdeI, HpaI, HindIII, AsuII and EcoRI) are situated within cI; hence insertion of foreign DNA into these sites causes depressed expression of the TcR gene from pR thus conferring the TcR phenotype on the harboring Escherichia coli strain. The use of pNS1 is facilitated by the presence of another selectable marker, ApR, its small size, and its known nucleotide sequence; no special host strain is required.

Base Sequence↗
PubMed1980

The hsd (host specificity) genes of E. coli K 12.

The hsd genes of E. coli K 12 have been cloned in phage lambda by a combination of in vitro and in vivo techniques. Three genes, whose products are required for K-specific restriction and modification, have been identified by complementation tests as hsdR, M and S. The order of these closely linked genes was established as R, M, S by analysis of the DNA of genetically characterised deletion derivatives of lambda hsd phages. The three genes are transcribed in the same direction but not necessarily as a single operon. Genetic evidence identifies two promoters, one from which transcription of hsdM and S is initiated and a second for the hsdR gene. The hsdR gene codes for a polypeptide of molecular weight approximately 130 000; hsdM for one of 62--65 000 and the hsdS gene was associated with two polypeptides of approximately 50 000. Circumstantial evidence suggest that one of these two polypeptides may be a degradation, or processed, derivative of the other. The hsdS polypeptide of E. coli B has a slightly higher mobility in an SDS-polyacrylamide gel than does that of E. coli K 12. A probe comprising most of the hsdR gene and all of the hsdM and S genes of E. coli K 12 shares extensive homology with the DNA of E. coli B but none with that of E. coli C.

Bacterial Proteins↗
PubMed1979

Restriction mapping of DNA of temperate Rhizobium meliloti phage 16-3: comparison of genetic and physical maps indicates a long, genetically silent chromosomal arm.

The complete restriction map of DNA (61.57 Kb) of temperate Rhizobium meliloti phage 16-3 has been constructed for enzymes BglII, HindIII, HpaI, KpnI, and a partial map for EcoRI. The strategy employed for mapping included the analysis of double, triple and partial digests; comparison of wild type and deletion mutants; and detailed analysis of subfragments, exploiting the presence of cohesive ends of the phage. Comparison of the genetic and physical maps indicates that one arm of the chromosome is genetically silent and/or contains nonessential genes.

Bacteriophages↗
PubMed1978

Physical mapping of bacteriophage lambda DNA with restriction endonuclease HpaI.

The restriction endonuclease from Haemophilus parainfluenzae, endoR.HpaI cleaves lambdacI857s7 DNA into 14 fragments. The sizes of these fragments were determined and a physical map was constructed. The ordering of the fragments was carried out using different deletion and substitution mutants of lambda phage, double cleavages with another restriction enzyme, endoR.BamHI, and partial protection of individual HpaI recognition sites by the antibiotics distamycin A and actinomycin D. HpaI produces fragments from the left arm of the lambda DNA genome, which may help in investigating the structure and function of this part of the phage.

Chromosome Mapping↗
PubMed1978

Cloning of an E. coli ribosomal RNA gene and its promoter region from lambdarifd18.

The DNA of the specialized transducing phage lambdarifd18, which carries a bacterial rRNA transcription unit, was digested with restriction enzymes EcoRI and/or BamHI. Attempts were made to clone fragments containing the presumed rRNA promoter region or the entire rRNA gene in RSF2124 or pBR313 plasmid vectors with the following results: (1) We failed to clone an EcoRI fragment with the rRNA promoter region in plasmid RSF2124. (2) A smaller EcoRI-BamHI fragment with the rRNA promoter was also unclonable by itself, but one recombinant was found containing this fragment together with another large (7 Mdaltons) fragment, derived from phage lambda. The presence of this large fragment proved to be essential. The identity of these DNA fragments in the recombinant clone was confirmed by redigestion with several restriction enzymes, hybridization with rRNA, and in vitro transcription experiments, which showed preferential rRNA transcription. (3) A BamHI fragment encompassing the entire rRNA gene was easily cloned. Such stable clones carried a doubled number of rRNA genes. In vitro transcription using the recombinant plasmid resulted in 70% rRNA transcription. These recombinant clones allow the easy purification of the relevant DNA fragments for further investigation including sequencing.

Chromosome Mapping↗
PubMed1977

A new sequence-specific endonuclease (Bsp) from Bacillus sphaericus.

A new restriction endonuclease has been isolated from Bacillus sphaericus R. The purification procedure includes Bio-Gel filtration, (NH4)2SO4 fractionation and phosphocellulose chromatography. After the phosphocellulose step the enzyme preparation is free of non-specific nucleases. Bsp cleaves double-stranded DNA with the same specificity as Bacillus subtilis (Bsu) and Haemophilus aegyptius (HaeIII) restriction endonucleases, as concluded from digests and double-digests of phiX174 replicative form DNA with Bsu and Bsp. The 5'-terminal nucleotide of the cleavage products was shown to be C. Bacillus sphaericus R produces Bsp in extremely large quantities and the enzyme can be easily purified in high yield.

Bacillus↗
PubMed1977

Protection of particular endonuclease R. Hind III cleavage sites by distamycin A, propyl-distamycin and netropsin.

It is shown that three related antibiotics, distamycin A, propyl-distamycin and netropsin, can protect certain endo R.Hind III cleavage sites from attack by endonuclease, giving rise, after endo R.Hind III digestion, to larger DNA fragments. Bacteriophage lambda DNA has six recognition sites for Hind III enzyme. Three of these sites: shind III 2, 3 and 6 can be protected from nuclease action by all the antibiotics used. Propyl-distamycin protects partly shind III 5, too. Netropsin protects partly sites shind III 5 and 4, while distamycin A protects all the sites but shind III 1 so the Hind III digestion produces only two large fragments of lambda DNA.

DNA Restriction Enzymes↗
PubMedDate not supplied

Cloning of the promoters of an Escherichia coli rRNA gene. New experimental system to study the regulation of rRNA transcription.

The promoters of the rrnB gene of Escherichia coli have been cloned on a multicopy, pBR322-derived plasmid by deleting most of the structural part of rrnB and fusing the terminators of the gene immediately to the promoters. Several further deletions were constructed to vary the promoter-terminator distance, destroy or damage selectively any of the promoters or terminators, and vary the distance between the two pairs of P1 P2 and P3 P4 promoters. All these transcription signals were shown to function on the plasmids in vitro and in vivo. The truncated in vivo transcription products initiated at the P1 and P2 promoters of the recombinant plasmids were found to be stable, and the accumulated transcripts could be easily distinguished from the chromosome-coded rRNA. This provides a convenient experimental system to study the regulation of rRNA biosynthesis.

Chromosome Deletion↗