Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 22
Filter
Add more filters










Publication year range
1.
Neurobiol Aging ; 26(5): 765-75, 2005 May.
Article in English | MEDLINE | ID: mdl-15708451

ABSTRACT

The gradual loss of striatal dopamine and dopaminergic neurons residing in the substantia nigra (SN) causes parkinsonism characterized by slow, halting movements, rigidity, and resting tremor when neuronal loss exceeds a threshold of approximately 80%. It is estimated that there is extensive compensation for several years prior to symptom onset, during which vulnerable neurons asynchronously die. Recent evidence would argue that much of the compensatory response of the nigrostriatal system is multimodal including both pre-synaptic and striatal mechanisms. Although parkinsonism may have multiple causes, the classic syndrome, Parkinson's disease (PD), is frequently modeled in small animals by repeated administration of the selective neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Because the MPTP model of PD recapitulates many of the known behavioral and pathological features of human PD, we asked whether the striatal cells of mice treated with MPTP in a semi-chronic paradigm enact a transcriptional program that would help elucidate the response to dopamine denervation. Our findings reveal a time-dependent dysregulation in the striatum of a set of genes whose products may impact both the viability and ability to communicate of dopamine neurons in the SN.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Corpus Striatum/drug effects , Gene Expression/drug effects , MPTP Poisoning/metabolism , Analysis of Variance , Animals , Corpus Striatum/metabolism , Disease Models, Animal , MPTP Poisoning/genetics , Male , Mice , Mice, Inbred C57BL , Models, Biological , Oligonucleotide Array Sequence Analysis/methods , Principal Component Analysis/methods , Reproducibility of Results
2.
Vaccine ; 23(7): 897-900, 2005 Jan 04.
Article in English | MEDLINE | ID: mdl-15603890

ABSTRACT

Purified Vero cell culture rabies vaccine "Abhayrab" manufactured by Human Biologicals Institute, Ooty, India was subjected for immunogenicity studies. Pre-exposure study was undertaken on 60 healthy volunteers (Group I) with vaccination on days 0, 7 and 21. A group of 75 patients of category II (Group II), 67 of category III (Group III) were given post-exposure prophylaxis and 88 patients of category III were administered with rabies immunoglobulins (Group IV) along with post-exposure prophylaxis as per World Health Organization (WHO) recommendations with a booster on day 90. The volunteers and patients vaccinated showed very few adverse side effects. The blood samples collected from volunteers (Group I) on days 14, 35 and 365 and patients (Group II-IV) on days 14, 30, 90 and 365 showed geometric mean titres (GMT) of >0.5 IU/ml. The study indicated new rabies vaccine manufactured in India was found to be safe and immunogenic.


Subject(s)
Rabies Vaccines/administration & dosage , Rabies Vaccines/immunology , Rabies/immunology , Rabies/prevention & control , Adult , Animals , Antibodies, Viral/biosynthesis , Antibodies, Viral/blood , Chlorocebus aethiops , Female , Humans , India , Injections, Intramuscular , Male , Middle Aged , Neutralization Tests , Rabies/blood , Rabies Vaccines/adverse effects , Rabies Vaccines/isolation & purification , Vero Cells
3.
Biotechniques ; Suppl: 44-7, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12083397

ABSTRACT

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.


Subject(s)
Bacillus Phages/genetics , DNA, Circular , DNA-Directed DNA Polymerase , Nucleic Acid Amplification Techniques/instrumentation , Sequence Analysis, DNA/instrumentation , Templates, Genetic , DNA, Bacterial/genetics , DNA, Viral , Escherichia coli/genetics , Escherichia coli/virology , Gene Library , Humans , Nucleic Acid Amplification Techniques/methods , Reproducibility of Results , Sensitivity and Specificity
4.
Proc Natl Acad Sci U S A ; 97(22): 11978-83, 2000 Oct 24.
Article in English | MEDLINE | ID: mdl-11035788

ABSTRACT

HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5' end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5' end of the RNA to make a secondary cut creating two products 8-9 nt long. RT then binds the new 5' end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3' end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.


Subject(s)
HIV Reverse Transcriptase/metabolism , Ribonuclease H/metabolism , Base Sequence , Hydrolysis , Molecular Sequence Data
5.
J Biol Chem ; 275(48): 37664-71, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-10956669

ABSTRACT

Synthesis of the minus strand of viral DNA by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase is accompanied by RNase H degradation of the viral RNA genome. RNA fragments remain after synthesis and are degraded by the polymerase-independent mode of RNase H cleavage. Recently, we showed that this mode of cleavage occurs by a specific ordered mechanism in which primary cuts are first, secondary and 5-nucleotide cuts are next, and second primary cuts occur last (Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P., J., and Bambara, R., A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 11978-11983). Ultimately the RNAs are cleaved into small fragments that can dissociate from the DNA template. Because the cleavage mechanism is an ordered series of events, we determined in this study whether any earlier cut is required for a later cut. By precisely inhibiting cleavage at each site, we examined the dependence of later cuts on cleavage at that site. We found that each cut is independent of the other cuts, demonstrating that the order of this stepwise mechanism is based on the rates of each cut. A mechanism for unlinked ordered cleavage consistent with these results is presented.


Subject(s)
HIV Reverse Transcriptase/metabolism , Ribonuclease H/metabolism , Base Sequence , Molecular Sequence Data , RNA/chemistry
6.
J Biol Chem ; 274(40): 28175-84, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10497170

ABSTRACT

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3'-ends of primers. Point mutations were created by alanine substitution at positions 224-235. Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A. Mutant W229A exhibited high fidelity and did not produce a dG:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions. This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT. We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses.


Subject(s)
DNA Replication , HIV Reverse Transcriptase/genetics , Base Pair Mismatch , DNA Primers , Mutagenesis , Templates, Genetic
7.
J Virol ; 73(7): 5803-13, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10364332

ABSTRACT

The nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) delavirdine (DLV) selects in vitro for the human immunodeficiency virus type 1 (HIV-1) RT mutation P236L, which confers high-level resistance to DLV but not other NNRTIs. Unexpectedly, P236L has developed infrequently in HIV-1 isolates obtained from patients receiving DLV; K103N is the predominant resistance mutation observed in that setting. We characterized the replication fitness of viruses derived from pNL4-3 containing P236L or K103N in both H9 and primary human peripheral blood mononuclear cell cultures infected in parallel with the two mutants. In the absence of DLV, p24 production by wild-type virus occurred more rapidly and to higher levels than with either mutant; P236L consistently demonstrated a two- to threefold decrease in p24 relative to K103N. At low levels of DLV, growth of wild-type virus was severely inhibited, and K103N replicated two- to threefold more efficiently than P236L. At high concentrations of DLV, P236L replication and K103N replication were both inhibited. Recombinant RTs containing K103N or P236L were analyzed for DNA polymerization on heteropolymeric RNA templates and RNase H degradation of RNA-DNA hybrids. Neither mutant demonstrated defects in polymerization. K103N demonstrated normal RNA 5'-end-directed RNase H cleavage and slowed DNA 3'-end-directed RNase H cleavage compared to wild-type RT. P236L demonstrated slowing of both DNA 3'-end- and RNA 5'-end-directed RNase H cleavage, consistent with its reduced replication efficiency relative to K103N. These data suggest that NNRTI resistance mutations can lead to reductions in the efficiency of RNase H cleavage, which may contribute to a reduction in the replication fitness of HIV-1.


Subject(s)
Anti-HIV Agents/pharmacology , DNA, Viral/metabolism , Defective Viruses/physiology , Delavirdine/pharmacology , HIV-1/physiology , RNA, Viral/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Ribonuclease H/metabolism , Virus Replication/drug effects , 5' Untranslated Regions , Cell Line , Defective Viruses/drug effects , Drug Resistance, Microbial , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , HeLa Cells , Humans , Kinetics , Mutagenesis
8.
J Biol Chem ; 273(7): 3808-16, 1998 Feb 13.
Article in English | MEDLINE | ID: mdl-9461561

ABSTRACT

Human immunodeficiency virus reverse transcribes its single-stranded RNA genome making a DNA copy. As synthesis proceeds, the RNA is simultaneously degraded to oligomers; one of these, the polypurine tract, primes synthesis of a plus strand DNA. The viral reverse transcriptase (RT) degrades all of the non-polypurine tract oligomers. We show that unlike other DNA polymerases the retroviral RT can bind either end of an annealed RNA primer, the 5'-end for degradation and the 3'-end for synthesis. The competition between the two binding modes at any primer determines whether it will be extended or degraded. The 5'-end binding can be suppressed in at least two ways. The sequence of the primer can be such that a region at the 5'-end is unannealed or a DNA primer can be annealed just adjacent to the 5'-end of the RNA primer. This promotes binding of RT to the RNA 3'-end, allowing a primer that would normally be degraded to be extended. Implications for human immunodeficiency virus replication and antiviral therapy are discussed.


Subject(s)
DNA/biosynthesis , HIV/enzymology , RNA-Directed DNA Polymerase/metabolism , Binding Sites , DNA Primers/genetics , Humans , Nucleic Acid Hybridization/genetics , RNA/chemistry , RNA/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease H/genetics , Ribonuclease H/metabolism
9.
J Biol Chem ; 272(27): 16769-77, 1997 Jul 04.
Article in English | MEDLINE | ID: mdl-9201981

ABSTRACT

We previously found that strand transfer by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is promoted at sites where RT pauses during synthesis. In this report, strand transfer is measured within the 5' transactivation response region (TAR) of HIV-1 RNA. We hypothesized that the stable hairpin structure of TAR would induce RT pausing, promoting RNase H-directed cleavage of the template and subsequent transfer at that site. We further predicted that HIV-1 nucleocapsid protein (NC), known to melt secondary structures, would decrease transfer. We show that TAR created a strong pause site for RT, but NC significantly promoted strand transfer. The effect of NC is specific, since other single strand binding proteins failed to stimulate transfer. In another unexpected outcome, preferred positions of internal transfer were not at the pause site but were in the upper stem and loop of TAR. Thus, we propose a new mechanism for transfer within TAR described by an interactive hairpin model, in which association between the donor and the acceptor templates within the TAR stem promotes transfer. The model is consistent with the observed stimulation of strand transfer by NC. The model is applicable to internal and replicative end transfer.


Subject(s)
HIV Long Terminal Repeat , HIV Reverse Transcriptase/metabolism , HIV-1/genetics , Transcriptional Activation , Base Sequence , Binding Sites , DNA, Viral/metabolism , Humans , Models, Genetic , Molecular Sequence Data , Nucleic Acid Conformation , Nucleocapsid/metabolism , Ribonuclease H/metabolism , Templates, Genetic
10.
J Biol Chem ; 272(23): 14523-31, 1997 Jun 06.
Article in English | MEDLINE | ID: mdl-9169409

ABSTRACT

Cellular tRNALys-3 serves as the primer for reverse transcription of human immunodeficiency virus, type 1 (HIV-1). tRNALys-3 interacts directly with HIV-1 reverse transcriptase, is packaged into viral particles and anneals to the primer-binding site (PBS) of the HIV-1 genome to initiate reverse transcription. Therefore, the priming step of reverse transcription is a potential target for antiviral strategies. We have developed a mutant tRNALys-3 derivative with mutations in the PBS-binding region such that priming specificity was re-directed to the highly conserved TAR stem-loop region. This mutant tRNA retains high-affinity binding to HIV-1 reverse transcriptase, viral encapsidation, and is able to prime at both the targeted TAR sequence and at the viral PBS. Constitutive expression of mutant tRNA in T-cells results in marked inhibition of HIV-1 replication, as determined by measurements of viral infectivity, syncytium formation, and p24 production. Inhibition of retroviral replication through interference with the normal process of priming constitutes a new anti-retroviral approach and also provides a novel tool for dissecting molecular aspects of priming.


Subject(s)
HIV-1/physiology , Virus Replication , Base Sequence , Cell Line , DNA Primers , Giant Cells , HIV Core Protein p24/biosynthesis , HIV Reverse Transcriptase/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis , Nucleic Acid Conformation , Polymerase Chain Reaction , RNA , T-Lymphocytes/virology , Transcription, Genetic , Transfection , Virion/physiology
11.
J Biol Chem ; 272(17): 11157-64, 1997 Apr 25.
Article in English | MEDLINE | ID: mdl-9111014

ABSTRACT

Human immunodeficiency virus (HIV) DNA synthesis is accompanied by degradation of genomic RNA by the RNase H of reverse transcriptase (RT). Two different modes of RNase H activity appear necessary for complete RNA removal. In one, occurring during minus strand synthesis, positioning of the RNase H is determined by binding of the polymerase active site to the DNA 3'-end. In the other, used for removal of remaining RNA fragments, positioning of RT for RNase H-directed cleavage is determined by the RNA 5'-ends. We attempted to identify RT amino acids responsible for these modes of positioning. Twelve RT mutants, each with one alanine replacement in residues 224 to 235, known as the primer grip region, were examined for catalytic abilities. Six of the examined primer grip mutants, although distant from the RNase H active site were altered in their ability to cleave RNA. The mutants P226A, F227A, G231A, Y232A, E233A, and H235A failed to perform RNA 5'-end-directed RNase H cleavage in heparin-challenged reactions. The last four mutants also lacked DNA synthesis and DNA 3'-end-directed RNase H cleavage activities in challenged reactions. Since mutants P226A and F227A carried out these latter reactions normally, these two residues specifically influence 5'-RNA-directed RNase H catalysis.


Subject(s)
HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , RNA, Viral/metabolism , Ribonuclease H/metabolism , Binding Sites/genetics , HIV Reverse Transcriptase/genetics , Mutagenesis, Site-Directed , Mutation , Protein Binding , Ribonuclease H/genetics , Structure-Activity Relationship , Substrate Specificity
12.
J Biol Chem ; 271(47): 29605-11, 1996 Nov 22.
Article in English | MEDLINE | ID: mdl-8939890

ABSTRACT

Two distinct plus strand initiation sites have been identified in human immunodeficiency virus (HIV), the central polypurine tract (cPPT) and the polypurine tract located just upstream of the U3 region (U3-PPT). When synthesis from the U3-PPT reaches the cPPT, the elongating primer causes limited strand displacement of the product created from the cPPT. We examined whether reverse transcriptase (RT) catalyzed strand transfer recombination is promoted by this process. Using a substrate having the viral sequence of the displaced region, we measured transfer of an elongating DNA primer from a donor DNA to an acceptor DNA. Strand transfer synthesis was only efficient when RT was performing strand displacement synthesis. Transfer efficiency was directly related to acceptor concentration but independent of the reaction time. Transfer could occur to acceptors containing 80, 40, or 20 nucleotides of homology with the template DNA. Using different acceptors, we found that DNA to DNA transfer occurred at positions throughout the donor template, except near the 5' end. This shows that a number of the sequences downstream of the cPPT region can promote transfer, but once synthesis has progressed to the point where the downstream segment is completely displaced transfer is not allowed. When the DNA to DNA transfer reactions were performed using a template containing nonviral sequences, the transfer efficiency dropped significantly. This indicates that transfer efficiency is determined by the sequences of the templates used. HIV-RT RNase H-dependent strand transfer between RNA templates is well documented. We propose a quite different mechanism for DNA to DNA transfer, consistent with the ability of RNase H minus RT to perform this reaction. If these DNA to DNA transfer events occur in vivo, they will result in plus strand recombination.


Subject(s)
DNA, Viral/genetics , HIV-1/genetics , DNA Primers , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Recombination, Genetic , Templates, Genetic
13.
J Biol Chem ; 271(37): 22331-8, 1996 Sep 13.
Article in English | MEDLINE | ID: mdl-8798393

ABSTRACT

Genome heterogeneity in retroviruses derives from poor fidelity of the reverse transcriptase (RT) and recombination via RT-catalyzed strand transfer synthesis. RTs lack proofreading ability, and they proficiently extend primers with mismatched termini. Recombination reactions carried out in vitro are accompanied by a high frequency of base substitution errors, suggesting a relationship. Here we provide evidence that misincorporation during RNA-directed DNA synthesis promotes strand transfer recombination. Experiments involved measurement of DNA synthesis, RNase H-directed cleavage, and strand transfer synthesis from preformed mismatched primers on RNA templates by human immunodeficiency virus (HIV) RT in vitro. A significant pause in synthesis occurred from a G(primer). rA(template) mismatch compared to the synthesis from a correctly paired (T.A) primer. The misincorporation-induced pause allowed an unusually large area of RT-RNase H-directed cleavage of the template RNA beneath the primer. Strand transfer to an acceptor molecule with sequence identical to the template RNA was about 50% more efficient than if the primer had had a correctly paired terminus. Overall transfer was measured over a large region of homology. Assuming that enhanced transfer occurs primarily at the site of the mismatch, the actual increase in transfer at that site must have been 1-2 orders of magnitude. Inclusion of a different acceptor molecule with complete complementarity to the originally mismatched 3' primer terminus resulted in an additional 2-fold increase in strand transfer efficiency. Overall, these results suggest the mechanism by which misincorporation during minus strand DNA synthesis in retroviral replication would promote high frequency recombination.


Subject(s)
HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , HIV-1/genetics , Leucine Zippers , Recombination, Genetic , DNA Primers/metabolism , DNA Replication , DNA, Viral/biosynthesis , Electrophoresis, Polyacrylamide Gel , Humans , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/metabolism , Templates, Genetic , Time Factors
14.
Nucleic Acids Res ; 24(9): 1710-8, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8649990

ABSTRACT

We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region. The resulting lacZalpha peptide fusion protein remained functional. Any frameshift mutations in the nef insert would put the downstream lacZ alpha peptide gene out of frame, eliminating alpha complementation. With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis. Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations. DNA-directed DNA synthesis generated frameshift mutations at a frequency approximately 10-fold higher than those arising from RNA-directed DNA synthesis. RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum. The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites. Results indicate that recombination is another source of mutations during viral replication.


Subject(s)
DNA Replication/genetics , Genes, nef/genetics , HIV-1/genetics , Mutagenesis/genetics , Virus Replication/genetics , Base Sequence , DNA Mutational Analysis , DNA, Recombinant/genetics , DNA, Viral/biosynthesis , Escherichia coli/genetics , Frameshift Mutation/genetics , Genetic Complementation Test/methods , HIV-1/physiology , Lac Operon/genetics , Molecular Sequence Data , RNA, Viral , Recombination, Genetic/genetics , Templates, Genetic
15.
J Biol Chem ; 271(4): 1966-71, 1996 Jan 26.
Article in English | MEDLINE | ID: mdl-8567645

ABSTRACT

According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo. We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer. The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation. Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not. The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis. Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA. This observation suggests that recombination is favored during strand displacement synthesis in vivo.


Subject(s)
DNA, Viral/biosynthesis , HIV Long Terminal Repeat/genetics , HIV-1/enzymology , RNA-Directed DNA Polymerase/metabolism , Base Sequence , DNA Primers/chemistry , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , HIV Reverse Transcriptase , HIV-1/genetics , Humans , Molecular Sequence Data , Templates, Genetic , Virus Replication
16.
J Biol Chem ; 271(4): 2063-70, 1996 Jan 26.
Article in English | MEDLINE | ID: mdl-8567660

ABSTRACT

RNA/DNA hybrids in human immunodeficiency virus (HIV) replication are cleaved by HIV-1 reverse transcriptase (RT) H in locations determined by hybrid structure. Minus strand DNA synthesis is accompanied by cleavage of template viral RNA directed by RT positioned at the growing 3' DNA end. Some RNA remains as oligomers annealed to the new DNA strand and is cut by RTs positioned at the 5' RNA ends. We constructed substrates to the test the hypothesis that internal helix structure, rather than strand end structure, drives the RT to position at 3' DNA and 5' RNA ends. On substrates with an RNA primer recessed on a DNA template, the 5' end of the RNA had a dominant role in the determination of RNase H cleavage positions. If the 5' end region of the RNA could not anneal, cleavage would not occur. Nevertheless, we obtained evidence that helix structure promotes the binding of RT to the end of the helical region closest to the 5' RNA/3' DNA end. When a DNA primer recessed on an RNA template had a 3' unannealed region, cleavage occurred, with RT positioned solely by helical structure at the 5' RNA/3' DNA end of the annealed region of the hybrid. Using substrates having RNA primers annealed to circular DNA templates, we showed that cleavage can be independent of the presence of a DNA 3'end and is directed by the 5' RNA end. Overall, the results suggest that the RT initially binds an internal region of the hybrid and then is driven in the direction to encounter a 3' DNA or 5' RNA end, where it is positioned for catalysts by the strand end. The requirement for two modes of RNA cleavage in viral replication and the unexpected requirement for the 5' RNA end structure are discussed.


Subject(s)
RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/metabolism , Base Sequence , DNA Primers/chemistry , DNA, Single-Stranded/metabolism , DNA, Viral/metabolism , HIV Reverse Transcriptase , Kinetics , Molecular Sequence Data , Nucleic Acid Hybridization , RNA, Viral/metabolism , RNA-Directed DNA Polymerase/chemistry , Recombinant Proteins , Ribonuclease H/chemistry , Structure-Activity Relationship , Substrate Specificity , Templates, Genetic
17.
Curr Microbiol ; 31(3): 193-8, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7663309

ABSTRACT

Glutamine synthetase (GS, EC 6.3.1.2) from Nocardia asteroides was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-150, and DEAE-Sepharose chromatography. The native molecular weight of the purified enzyme was determined to be 720 kDa. SDS-PAGE analysis of the purified preparation revealed a single band corresponding to 59 kDa, indicating the possible presence of 12 identical subunits. The divalent cations Mn2+ and Mg2+ were found to be essential for optimal transferase and biosynthetic activity, respectively. The optimal pH and temperature for both activities of the enzyme were found to be 7.2 and 50 degrees C. Amino acids such as L-alanine, glycine, and aspartate inhibited the GS activity. The Km values for the substrates of the biosynthetic reaction ATP, glutamate, and ammonium chloride were found to be 400 microM, 7.7 mM, and 200 microM, respectively. Addition of ammonium chloride to the nitrogen-limited culture resulted in a decrease of GS transferase and biosynthetic activities. Phosphodiesterase treatment of the extract from ammonia-shocked cultures showed an increase in GS transferase activity. The results indicate the possible regulation of GS by covalent modification.


Subject(s)
Glutamate-Ammonia Ligase/isolation & purification , Glutamate-Ammonia Ligase/metabolism , Nocardia asteroides/enzymology , Amino Acids/pharmacology , Ammonium Chloride/pharmacology , Glutamate-Ammonia Ligase/antagonists & inhibitors , Glutamate-Ammonia Ligase/chemistry , Hydrogen-Ion Concentration , Kinetics , Magnesium , Manganese , Molecular Weight , Temperature
18.
J Biol Chem ; 270(9): 4861-9, 1995 Mar 03.
Article in English | MEDLINE | ID: mdl-7533167

ABSTRACT

The action of the dipyridodiazepinone nevirapine (BI-RG-587) on polymerization and RNase H activities of human immunodeficiency virus reverse transcriptase (RT) was examined. Substrates using heteropolymeric DNA primers hybridized to complementary RNA templates were employed. Challenged assays were performed that allowed measurement of activity of the RT resulting from a single round of binding of RT to substrate. Results demonstrated that nevirapine alters the cleavage specificity of the RNase H. Instead of a primary cleavage approximately 18 nucleotides upstream of the DNA 3' terminus, multiple cleavages were observed ahead of and behind this site. This indicated that the compound facilitates sliding of the RT away from the DNA primer terminus allowing cleavage at more sites. The change in specificity occurred whether the primer terminus was at the end or internal on the template. Experiments with RNA primers on circular DNA demonstrated a nevirapine-induced stimulation of RNase H activity beyond the increase expected from the change in cleavage specificity. Examination of polymerization showed that the compound decreased both the number of primers that underwent synthesis and the processive elongation of those primers. The significance of these results with respect to viral replication and recombination is discussed.


Subject(s)
HIV-1/enzymology , Pyridines/pharmacology , RNA-Directed DNA Polymerase/drug effects , Ribonuclease H/metabolism , Base Sequence , DNA Primers , HIV Reverse Transcriptase , Hydrolysis , Kinetics , Molecular Sequence Data , Nevirapine , RNA-Directed DNA Polymerase/metabolism , Reverse Transcriptase Inhibitors , Substrate Specificity , Templates, Genetic
19.
J Bacteriol ; 176(9): 2648-53, 1994 May.
Article in English | MEDLINE | ID: mdl-8169214

ABSTRACT

The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacillus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) synthase-alpha-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed E1, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the alpha-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate alpha-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity.


Subject(s)
Bacillus subtilis/metabolism , Ketoglutarate Dehydrogenase Complex/metabolism , Phenylbutyrates/metabolism , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Chorismic Acid/metabolism , Cyclohexanes , Cyclohexenes , Ketoglutarate Dehydrogenase Complex/drug effects , Mutation , Oxo-Acid-Lyases/biosynthesis , Salicylates/metabolism , Subcellular Fractions/enzymology , Succinates/metabolism , Vitamin K/pharmacology
20.
Mycopathologia ; 124(2): 69-72, 1993 Nov.
Article in English | MEDLINE | ID: mdl-7911977

ABSTRACT

Specific enzymes of ammonium assimilation were measured in cell-free extracts of Nocardia asteroides grown in a synthetic medium with glutamate as the nitrogen source. Cell-free extracts had active glutamine synthetase (GS) and glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) but glutamate dehydrogenase (GDH) could not be detected in the enzyme preparation. This shows that GS/GOGAT is the major pathway of ammonium assimilation in N. asteroides.


Subject(s)
Glutamates/metabolism , Nocardia asteroides/metabolism , Quaternary Ammonium Compounds/metabolism , Alanine Dehydrogenase , Amino Acid Oxidoreductases/analysis , Glucose/metabolism , Glutamate Dehydrogenase/analysis , Glutamate Synthase/analysis , Glutamic Acid , Nocardia asteroides/enzymology , Nocardia asteroides/growth & development
SELECTION OF CITATIONS
SEARCH DETAIL
...