Interaction of Cibacron Blue F3GA with Escherichia coli DNA polymerase I and with T4 DNA polymerase.

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.

Escherichia coli aminodeoxychorismate synthase: analysis of pabB mutations affecting catalysis and subunit association.

p-Aminobenzoic acid (PABA), an essential component of the vitamin folic acid, is derived from the aromatic branch-point precursor chorismate in two steps. 4-Amino-4-deoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate, and is composed of two subunits, PabA and PabB. While various experiments have suggested that PabA and PabB act as a complex, attempts to isolate the intact complex have failed. We report here the first successful copurification of PabA and PabB by gel filtration chromatography. The association of PabA and PabB is greatly enhanced by the presence of 5 mM glutamine, and by preincubation at 37 degrees C. Conversely, the association is greatly reduced at cold temperatures. We also report the isolation and characterization of both chemically induced and site-directed mutations in PabB. Mutated PabB enzymes fall into three categories according to their properties: deficiency of chorismate amination coupled with failure to associate with PabA, deficiency of chorismate amination coupled with retention of PabA association, and competency of chorismate amination with failure of PabA association.

A rapid purification of T4 polynucleotide kinase using Blue Dextran-Sepharose chromatography.

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.

Nucleotide sequence of Escherichia coli pabA and its evolutionary relationship to trp(G)D.

We have determined the entire nucleotide sequence of Escherichia coli pabA. A comparison of the nucleotide and amino acid sequences of pabA and trp(G) D reveals extensive homology, suggesting that these two genes arose from a common ancestor. pabA and trp(G) D are 44% homologous at the amino acid level and 53% homologous at the nucleotide sequence level. The nucleotide sequences can be divided into regions of high homology, in which most nucleotide changes occur in the third position of codons and do not effect the amino acid sequence, and regions which show almost no DNA homology. Divergence in these non-homologous regions appears to have resulted from single-base substitutions as well as the rearrangement of small regions of DNA by inversion, deletion and duplication.

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens.

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.

DNA sequence analysis of prm-mutations of coliphage lambda.

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.

Effects of scopolamine and hippocampal lesions on negative patterning discrimination performance in rats.

Rats were trained in operant chambers to perform an appetitive negative patterning successive discrimination. They were required to respond to the left in response to a tone or click and right to a tone-click compound. Scopolamine and methyl scopolamine impaired performance accuracy and increased response latency and response omissions. Subsequent hippocampal aspiration lesions initially impaired accuracy, which later improved. Lesions decreased response latencies. Finally, the effects of scopolamine and methyl scopolamine were shown to be similar in lesioned and control rats, suggesting that the hippocampus is not involved in the actions of these drugs on this task.

Characterization of a mutationally altered dihydropteroate synthase contributing to sulfathiazole resistance in Escherichia coli.

A series of Escherichia coli strains were selected for increasing resistance to sulfathiazole. Resistance occurred in seven increments, suggesting the accumulation of several mutations that contributed to overall sulfathiazole resistance. All of the resistant strains had a sulfathiazole-resistant dihydropteroate synthase with a Pro to Ser substitution at amino acid position 64. Overproduction of the wild-type enzyme did not result in sulfathiazole resistance, however overproduction of the mutant enzyme resulted in significant resistance. Conversely, overproduction of the wild-type enzyme in a sulfathiazole-resistant background resulted in a decrease in resistance. Although the specific activity of DHPS in crude extracts was not significantly different from the wild type, the amino acid substitution resulted in an enzyme with a tenfold increase in the Km for p-aminobenzoate, and a 100-fold increase in the Ki for sulfathiazole.

Characterization and sequence of the Escherichia coli panBCD gene cluster.

A 4589 bp DNA segment containing the Escherichia coli panBCD gene cluster was sequenced, and found to contain 6 complete open reading frames. panB, panC, and panD were identified by subcloning and insertional mutagenesis. The orientation of panD was also confirmed by orientation-specific expression of asparate-1-decarboxylase. panB and panC lie adjacent to one another, but are separated from panD by orf3, which is oriented in the opposite direction. Interruptions in the remaining open reading frames did not affect growth on glucose-minimal medium. No significant similarity to sequences in databases was found for orf1 and orf2. Orf3 contained extensive similarity to reading frames defined by E. coli yjiP, yjiQ, yhgA, and yafD. The function of these amino acid sequences is as yet undefined.

p-Aminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC.

p-Aminobenzoate, a component of the vitamin folate, is one of seven compounds derived from the aromatic precursor chorismate in Escherichia coli. Historically the gene products of pabA and pabB were assumed to be sufficient for de novo p-aminobenzoate biosynthesis. Recent studies, however, have shown that these proteins, as nonidentical subunits of a single enzyme, act on chorismate to form a diffusible intermediate, most likely 4-amino-4-deoxychorismate. This intermediate is then converted to p-aminodeoxychorismate lyase (Nichols, B. P., Seibold, A. S., and Doktor, S. Z. (1989) J. Biol. Chem. 264, 8597-8601). Here we describe partial characterization of the intermediate and the purification of aminodeoxychorismate lyase 4100-fold to near homogeneity. Further purification of this enzyme by high pressure liquid chromatography permitted isolation of a pure sample that yielded N-terminal sequence. A 64-fold redundant oligonucleotide probe was used to identify a lambda clone containing the gene encoding aminodeoxychorismate lyase. The aminodeoxychorismate lyase gene, designated pabC, was mapped to 25 min on the E. coli chromosome and lies on a 7.5-kilobase pair EcoRI fragment. A strain harboring a pACYC184 recombinant containing pabC overproduced aminodeoxychorismate lyase activity 77-fold.

Expression of Escherichia coli pabA.

Escherichia coli pabA encodes the glutamine amidotransferase subunit of p-aminobenzoate synthase. p-Aminobenzoate synthase catalyzes the conversion of chorismate and glutamine to 4-amino-4-deoxychorismate, which is then converted to p-aminobenzoate by a 4-amino-4-deoxychorismate lyase. The 5'-terminal segment of pabA was previously shown to be transcribed from two different promoters, one near the pabA coding sequence (P1) and one preceding fic (P2). However, a pabA-lacZ translational fusion was expressed only from the mRNA originating at P1. We have determined that expression of a pabA-lacZ chromosomal fusion is not changed by p-aminobenzoate limitation, growth rate, catabolite repression, overexpression of either p-aminobenzoate synthase subunit, or gene dosage of pabA and pabB. The lack of pabA expression from P2 appears to be the result of a stable secondary structure in the intergenic space preceding pabA that sequesters the pabA ribosome binding site. Disruption of the secondary structure by mutation allowed expression of pabA from P2, as did translation of ribosomes into the fic-pabA intergenic region.

Mapping and cloning of Eco RI-fragments of bacteriophage T5+ DNA.

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.

Nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli: an evolutionary comparison.

The complete nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli were determined. The nucleotide sequences are 24.8% divergent, compared with amino acid sequence divergence of 14.9%. Over half of the codons of each gene contain synonymous nucleotide changes. The pattern of synonymous nucleotide changes is consistent with the interpretation that such changes result from random mutational events. We do not find any evidence indicating that codon selection or RNA structure is of major selective value. We conclude that polypeptide function is the primary basis of selection in trpA and that most synonymous codon changes are selectively neutral.

178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA.

A nucleotide sequence of 61 nucleotides at the left end and 117 nucleotides at the right end of DNA from bacteriophage lambdacI857Sam7 was determined by the Maxam and Gilbert method. A perfect inverted repeat sequence of 10 nucleotides is near the left end, and one of 15 nucleotides is near the right end. DNA from another closely related lambda strain, lambdacI857prm116Sam7, has about 10% divergence in the sequence of the first 110 nucleotides at the right end and has a 17-member perfect inverted repeat sequence.

Sequence analysis of the nicks and termini of bacteriophage T5 DNA.

Bacteriophage T5 DNA, when isolated from mature phage particles, contains several nicks in one of the two strands. The 5'-terminal nucleotides at the nicks were labeled with polynucleotide kinase and [gamma-32P]ATP, and the 3'-terminal nucleotides were labeled with Escherichia coli DNA polymerase I and [alpha-32P]dGTP. The sequences around the nicks were analyzed by partial nuclease digestion followed by homochromatography fractionation of the resulting oligonucleotides. The nicks had at least the sequence -PuOH pGpCpGpC- in common. In addition, the two 5' external termini had the first seven nucleotides in common.

Gene amplification contributes to sulfonamide resistance in Escherichia coli.

A sulfathiazole-resistant strain of Escherichia coli was isolated and shown to contain a fourfold tandemly amplified segment of DNA 18 kilobase pairs in length in addition to a mutationally altered dihydropteroate synthase, the target enzyme for sulfonamide inhibition. The amplified DNA contained a gene designated sur that contributed to sulfathiazole resistance when present in greater amounts than those in the wild type. Sulfathiazole resistance was markedly decreased upon loss of the amplified DNA after nonselective growth. Plasmids that contained sur also conferred only weak sulfathiazole resistance on wild-type strains. Comparison of the restriction maps of the amplified DNA, wild-type DNA, and sur-containing plasmids showed that a DNA rearrangement occurred before or concomitant with the DNA amplification event. The DNA rearrangement resulted from an IS5 insertion, which, in conjunction with an IS5 element residing near sur in the wild-type strain, resulted in an -IS5-sur-IS5- configuration. Homologous recombination could account for duplication and subsequent amplification of the sur region. High-copy-number plasmids containing the sur locus did not express a sulfathiazole-resistant dihydropteroate synthase, nor did they overexpress wild-type dihydropteroate synthase. These data suggest that the high level of sulfathiazole resistance in this strain results from a synergistic effect of two different mutations.