The chromosomal insertion sites of Tn10-containing Escherichia coli strains were amplified by inv... more The chromosomal insertion sites of Tn10-containing Escherichia coli strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined. In 95 strains analyzed, 88 unique Tn10 positions were determined and matched to the E. coli chromosome sequence. Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots RhsA and RhsB.
The Eco RI-fragments of bacteriophage T5 DNA were mapped using a technique which involves primari... more The Eco RI-fragments of bacteriophage T5 DNA were mapped using a technique which involves primarily length measurements of molecules observed in the electron microscope. Since Eco RI cleavage generates termini with 4-nucleotide long cohesive ends, fragments of complete and partial Eco RI digests were covalently circularized with DNA ligase at dilute DNA concentrations before measuring relative to internal length standards. This established the order of the internal Eco RI fragments. The two external Eco RI fragments, which had only one Eco RI terminus, were positioned relative to the internal fragments by identifying the location of some of the naturally-occurring nicks in partially denatured linear Eco RI fragments. An attempt was made to clone each of the internal Eco RI-fragments of T5 DNA via transformation into E. coli after ligation in vitro with the plasmid pMB 9. Only one fragment could be cloned and this fragment did not specify any new polypeptides in minicells of either the E. coli EK1 host, X1411, or the EK 2 host, X1776.
Individual rapid procedures for the enrichment of Escherichia coli DNA polymerase I and of bacter... more Individual rapid procedures for the enrichment of Escherichia coli DNA polymerase I and of bacteriophage T4 DNA polymerase free of endonuclease activity are described using Blue dextran-Sepharose chromatography. The blue dye of Blue dextran-Sepharose selectively binds to the deoxynucleoside triphosphate substrate site of the E. coli but not the T4 enzyme indicating that the catalytic sites of these two enzymes which catalyze the same polymerization reaction in vitro are quite distinct.
A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dep... more A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.
1. Virology. 1977 Dec;83(2):396-403. The nucleotide sequence at the 3'-termini of three majo... more 1. Virology. 1977 Dec;83(2):396-403. The nucleotide sequence at the 3'-termini of three major T5 DNA fragments. Nichols BP, Donelson JE. PMID: 929984 [PubMed - indexed for MEDLINE]. Publication Types: Research Support ...
The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compound... more The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.
Nucleotide sequence changes associated with mutation of the prm promoter of bacteriophage lambda ... more Nucleotide sequence changes associated with mutation of the prm promoter of bacteriophage lambda have been determined. Prm-mutations have been assigned to two classes. Class I mutations appear to affect the interaction of RNA polymerase with prm; six class I mutations affect four sites, located 14, 33, 38, and 39 bp preceding the prm transcription startpoint. Class II mutations appear to owe their Prm-phenotype to a change in OR, which could prevent activation of prm by repressor. All three class II mutations are in OR 1.
The chromosomal insertion sites of Tn10-containing Escherichia coli strains were amplified by inv... more The chromosomal insertion sites of Tn10-containing Escherichia coli strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined. In 95 strains analyzed, 88 unique Tn10 positions were determined and matched to the E. coli chromosome sequence. Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots RhsA and RhsB.
The Eco RI-fragments of bacteriophage T5 DNA were mapped using a technique which involves primari... more The Eco RI-fragments of bacteriophage T5 DNA were mapped using a technique which involves primarily length measurements of molecules observed in the electron microscope. Since Eco RI cleavage generates termini with 4-nucleotide long cohesive ends, fragments of complete and partial Eco RI digests were covalently circularized with DNA ligase at dilute DNA concentrations before measuring relative to internal length standards. This established the order of the internal Eco RI fragments. The two external Eco RI fragments, which had only one Eco RI terminus, were positioned relative to the internal fragments by identifying the location of some of the naturally-occurring nicks in partially denatured linear Eco RI fragments. An attempt was made to clone each of the internal Eco RI-fragments of T5 DNA via transformation into E. coli after ligation in vitro with the plasmid pMB 9. Only one fragment could be cloned and this fragment did not specify any new polypeptides in minicells of either the E. coli EK1 host, X1411, or the EK 2 host, X1776.
Individual rapid procedures for the enrichment of Escherichia coli DNA polymerase I and of bacter... more Individual rapid procedures for the enrichment of Escherichia coli DNA polymerase I and of bacteriophage T4 DNA polymerase free of endonuclease activity are described using Blue dextran-Sepharose chromatography. The blue dye of Blue dextran-Sepharose selectively binds to the deoxynucleoside triphosphate substrate site of the E. coli but not the T4 enzyme indicating that the catalytic sites of these two enzymes which catalyze the same polymerization reaction in vitro are quite distinct.
A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dep... more A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.
1. Virology. 1977 Dec;83(2):396-403. The nucleotide sequence at the 3'-termini of three majo... more 1. Virology. 1977 Dec;83(2):396-403. The nucleotide sequence at the 3'-termini of three major T5 DNA fragments. Nichols BP, Donelson JE. PMID: 929984 [PubMed - indexed for MEDLINE]. Publication Types: Research Support ...
The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compound... more The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.
Nucleotide sequence changes associated with mutation of the prm promoter of bacteriophage lambda ... more Nucleotide sequence changes associated with mutation of the prm promoter of bacteriophage lambda have been determined. Prm-mutations have been assigned to two classes. Class I mutations appear to affect the interaction of RNA polymerase with prm; six class I mutations affect four sites, located 14, 33, 38, and 39 bp preceding the prm transcription startpoint. Class II mutations appear to owe their Prm-phenotype to a change in OR, which could prevent activation of prm by repressor. All three class II mutations are in OR 1.
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Papers by Brian Nichols