Biomedical subjects

D K Dube

Publications and source records attributed to D K Dube.

At least 55 records · Page 3Linked to original sources

PubMed1992

Permissible amino acid substitutions within the putative nucleoside binding site of herpes simplex virus type 1 encoded thymidine kinase established by random sequence mutagenesis [corrected].

We determined the essentiality of all amino acid replacements within an 11-codon sequence in the putative nucleoside-binding site of thymidine kinase encoded by herpes simplex virus type 1. This involved partial randomization of 11 codons in the gene to create a degenerate library, followed by genetic complementation using a tk- Escherichia coli strain and selection of unnatural active enzymes. We produced and tested 53,000 variants; of which 190 were found to be biologically active. Sequence analyses of functional variants revealed a high degree of flexibility in accommodating different types of amino acid substitutions in this region. However, no replacement was tolerated at proline-173, whereas tyrosine-172 could be replaced by only phenylalanine. To further define permissible substitutions at specified positions, we constructed a library with randomization at only four test codons. We produced and tested 600,000 variants; of which only 5 were active. Again proline-173 was conserved, and only tyrosine and phenylalanine were found at position 172. The identification of these conserved amino acids should provide important insights into the understanding of the structural basis of catalysis by this enzyme.

Amino Acid Sequence↗

PubMed1992

LTR sequence and phylogenetic analyses of a newly discovered variant of HTLV-I isolated from the Hagahai of Papua New Guinea.

A 631-bp region of the long terminal repeat (LTR) of a variant of human T-cell lymphoma/leukemia virus type I (HTLV-I), isolated from a healthy member of a remote, recently contacted group (Hagahai) in Papua New Guinea, was sequenced and compared to LTR sequences of other members of the primate T-cell lymphoma virus group (PTLV), including HTLV-I, simian T-cell lymphoma virus (STLV-I) and HTLV-II. Sequence analysis of the LTR of this New Guinean isolate, designated as HTLV-I(PNG-1), indicated a sequence divergence of 8.4% to 10.4% from prototype Japanese HTLV-I(ATK) and other HTLV-I and STLV-I isolates and 48.6% diversity from HTLV-II. Few mutations were found in the core elements of the transcriptional enhancer regions, the TATA box promoter, and the polyadenylation signal and site. Further, the observed changes did not significantly alter the inferred stability of the Rex response element, a stem loop structure critical for polyadenylation and Rex protein binding. Dendograms based on LTR sequences indicated that the strain of virus that evolved into HTLV-I(PNG-1) diverged from the other PTLV in the distant past, just after the progenitors of STLV-I from Asia, but before the ancestors of STLV-I from Africa. By contrast, other HTLV-I isolates were found to represent strains of virus that have diverged more recently and clustered primarily according to their geographical origin. These data confirm that HTLV-I(PNG-1) is a new and distinct variant of the PTLV group. Also, our analyses suggest that both HTLV-I and STLV-I may have originated in the Indo-Malay region and eventually spread to Africa and then to the New World and Japan with horizontal transmission between man and nonhuman primates possibly occurring over thousands of years.

Base Sequence↗

PubMed1992

Evolutionary insights on the origin of human T-cell lymphoma/leukemia virus type I (HTLV-I) derived from sequence analysis of a new HTLV-I variant from Papua New Guinea.

Recent studies have established the presence of human T-cell lymphoma/leukemia virus type I (HTLV-I) in Melanesia. An HTLV-I strain, PNG-1, has now been isolated from a healthy member of the Hagahai, a remote, recently contacted group in Papua New Guinea. To further characterize PNG-1, we employed polymerase chain reaction amplification with subsequent cloning and sequencing of amplified products. Sequence analyses of amplified regions of pol, env, and pX genes of this variant indicate marked heterogeneity (approximately 7%) from prototype HTLV-I. Based on available sequence data, PNG-1 is distinct from all other known HTLV-I strains and diverges from the common ancestor of HTLV-I prior to prototype isolates. The data also suggest, therefore, that HTLV-I originated in the Indo-Malay region rather than Africa.

Amino Acid Sequence↗

PubMed1991

Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.

We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

The association of thymidine kinase activity and thymidine transport in Escherichia coli.

We have constructed a series of mutants within the putative nucleoside-binding site of the herpes simplex type-1 virus (HSV-1) thymidine kinase (TK)-encoding gene (tk), contained within an expression vector. While most mutations within this sequence produce an inactive protein, we find no absolute requirement for the wild-type Ile166 and Ala167. The uptake of thymidine (dT) into Escherichia coli tdk-, lacking functional endogenous TK activity, is proportional to the amount of TK activity expressed from the heterologous HSV-1 tk gene. In contrast, there is no enhancement in deoxycytidine uptake into E. coli producing (HSV-1) TK. These results imply a specific role for TK in the active transport of dT into E. coli.

Amino Acid Sequence↗

PubMed1989

Sequence specificity of pausing by DNA polymerases.

We have constructed recombinant M13 DNA templates containing stretches of oligo (purines) and oligo (pyrimidines). Each of these inserts hinders the advancement of the large fragment of E. coli Pol I during DNA synthesis. The pattern of blockage is independent of changes in KCl or Mg2+ concentrations and pausing is moderately alleviated at lower pH. Blockage is not affected by either the concentration of template or by the position of the DNA primer. The pattern of pause sites is similar for calf thymus DNA polymerase-alpha, implying that replicative barriers are determined by the structure of the DNA at its growing point. There is a lack of correlation between the position of pause sites with different inserts and known alternate DNA structures. Thus, the homo-oligomeric inserts may possess a different structure when complexed with DNA polymerase. This concept accounts for the appearance of unique new upstream and downstream pause sites that result from the insertion of each oligonucleotide.

Base Sequence↗

PubMed1989

Mutants generated by the insertion of random oligonucleotides into the active site of the beta-lactamase gene.

We have remodeled the gene coding for beta-lactamase by replacing DNA at the active site with random nucleotide sequences. The oligonucleotide replacement (Phe66XXXSer70XXLys73) preserves the codon for the active serine-70 but also contains 15 base pairs of chemically synthesized random sequences that code for 2.5 x 10(6) amino acid substitutions. From a population of Escherichia coli infected with plasmids containing these random inserts, we have selected seven new active-site mutants that render E. coli resistant to carbenicillin and a series of related analogues. Each of the new mutants contains multiple nucleotide substitutions that code for different amino acids surrounding serine-70. Each of the mutants exhibits a temperature-sensitive beta-lactamase activity. This technique offers the possibility of constructing alternative active sites in enzymes on the basis of biological selection for functional variants.

Amino Acid Sequence↗

PubMed1989

Evidence for a chromosomally determined bacteriocin production by Klebsiella pneumoniae.

Location of the genes responsible for pneumocin production in Klebsiella pneumoniae was examined by classical procedures. Conjugal intrageneric transfers, elimination experiments with various curing agents, high temperature and plasmid isolation procedures showed that this strain did not harbour any plasmid. Hence chromosomal location of the genetic determinants is suggested.

Bacteriocins↗

PubMed1989

Selection of new biological activities from random nucleotide sequences: evolutionary and practical considerations.

Recent advances in the selection of biologically active DNA sequences from random populations are reviewed. Within the framework of evolution, forces are considered that have precluded the testing of all possible DNA sequences, purely with regard to their functionality as genetic regulatory elements or protein coding sequences. Examples are drawn from cassette mutagenesis of enzyme active sites, protein domain replacement by fusion with random genomic digests, and the selection of bacterial promoters from random DNA. Efforts to derive new activities are examined, and the likelihood of future success is evaluated.

Base Sequence↗

PubMed1989

Role of liver and brain lysosomal and mitochondrial enzymes in development of delayed neuropathy in hens by 0,0-diisopropyl phosphorofluoridate.

Subacute dose of 0,0-diisopropyl phosphorofluoridate (DFP), a potent organophosphorus ester capable of producing delayed neurotoxicity (OPIDN), did not produce any significant change in the levels of lysosomal and mitochondrial marker enzymes of brain, liver and serum at any time after treatment in hens protected with atropine. The results suggest the absence of any involvement of mitochondrial and lysosomal enzymes at any stage in the development of OPIDN in susceptible species by treating with DFP.

Animals↗

PubMed1986

Rearrangements of DNA mediated by terminal transferase.

To assess the involvement of terminal transferase in generating immunoglobulin diversity, the mutagenic potential of this enzyme has been measured. The frequency of single base substitutions during copying of phi X174 DNA by DNA polymerase beta is increased by, at most, 3-fold upon the addition of terminal transferase. However, terminal transferase is highly mutagenic, either alone or with DNA polymerase beta, in a forward mutation assay using M13mp2 DNA. The frequency of complex mutants, as determined by DNA sequence, is increased by greater than 100-fold. These mutants involve the deletion of a variable number of bases initially present in the template sequence and the addition of a sequence of nucleotides rich in guanine residues. Analysis of these mutants suggests an antibody diversity model implicating terminal transferase in the imprecise linkage of variable, joining, and diversity segments during the formation of functional immunoglobulin genes.

Animals↗

PubMed1984

Assessment of the carcinogenic potential of a proposed food coloring additive, laccaic acid, using short-term assays.

Laccaic acid is a red colored natural dye produced by the insect Laccifer lacca or Coccus lacca. It is obtained in large amounts as a by-product of the shellac industry and has been considered for general use as a food coloring agent. Laccaic acid is found to have no mutagenic activity as assessed by two short-term assays: the Salmonella/microsome mutagenicity test, and the phi X fidelity assay. However, laccaic acid did inhibit metabolic cooperation in Chinese hamster V79 cells. These results suggest that laccaic acid should be tested in animals with particular emphasis on in vivo models for tumor promotion.

Animals↗

PubMed1983

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.

Animals↗

PubMed1983

Antitrypanosomal activity of sinefungin.

Sinefungin, a naturally occurring antifungal antibiotic nucleoside containing an ornithine residue, linked by a C-C bond to C-5' of adenosine, cures mice infected with Trypanosoma brucei brucei, T. congolense, or T. vivax; the effect of the drug is more pronounced towards T. congolense. Anti-trypanosomal activity of sinefungin could be the result of the inhibition of transmethylation reactions or of polyamine biosynthesis--or both--in parasites.

Adenosine↗

PubMed1982