PubMed Health⌕ Search

Biomedical subjects

L Tarrago-Litvak

Publications and source records attributed to L Tarrago-Litvak.

At least 55 records · Page 3Linked to original sources
PubMed1990

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys).

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.

Base Sequence↗
PubMed1989

Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys.

Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene. The previously described purification procedure for the yeast-expressed reverse transcriptase [Barr, P.J., Power, M.D., Chun Ting Lee-Ng, Gibson, H. & Luciw, P. (1987) Bio/Technology 5, 486-489] has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates.

Animals↗
PubMed1988

Purification, immunological and biochemical characterization of Ap4A binding protein from Xenopus laevis oocytes.

Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) binding protein specifically binds Ap4A. The protein has been purified from Xenopus laevis oocytes and presents an estimated molecular weight of 100,000 by gel filtration. In the first stages of the purification, the Ap4A binding activity is found associated to DNA polymerase alpha-DNA primase, forming heterogeneous high molecular weight complexes. A monoclonal antibody has been prepared against the purified Ap4A binding protein. The antibody partially neutralizes the Ap4A binding activity. Using the immunoblot technique, it has been shown that the antibody is able to recognize either native or SDS-denatured Ap4A binding protein. The monoclonal antibody immunoreacted with a polypeptide of 90,000 which coincides with the molecular weight obtained by gel chromatography and indicates that the native Ap4A binding protein from Xenopus oocytes is probably a monomeric protein.

Adenine Nucleotides↗
PubMed1986

Studies on DNA polymerases of Xenopus laevis oocytes: subcellular distribution and physical association of DNA polymerase alpha inside the nucleus.

The localization of DNA polymerases in Xenopus laevis oocytes and eggs was studied. Oocytes have a high level of DNA polymerase alpha activity detected exclusively in the nuclei, while mitochondria contain all the DNA polymerase activity of the gamma type. No DNA polymerase beta activity is present in the nuclear fraction. Moreover, the beta activity is not present in unfertilized eggs. In oocytes, DNA polymerase alpha is weakly bound to the nucleoplasm. The leakage of the enzyme from whole nuclei can be prevented using polyvinylpyrrolidone, a nuclear pore sealing agent.

Animals↗
PubMed1985

2'-Fluoro-2'-deoxycytidine triphosphate as a substrate for RNA- and DNA-dependent DNA polymerases.

The ability of the analog 2'-fluoro-2'-deoxycytidine triphosphate (dCflTP) to be used as a substrate in the reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha and AMV reverse transcriptase has been studied. The apparent Km values for dCTP and dCflTP, using activated DNA as templates, were 0.6 microM and 7 mM with DNA polymerase alpha and 0.14 microM and 7 microM with AMV reverse transcriptase, respectively. As observed with dCTP, aphidicolin was a noncompetitive inhibitor in the DNA polymerase alpha-catalyzed DNA synthesis; the Ki values were about 2 microM for both substrates. dCflTP can also be incorporated into DNA synthetized by other eukaryotic DNA polymerases and by reverse transcriptase with RNA as a template, both in the presence or absence of (dT)12 primer.

Animals↗
PubMed1985

2'-Fluoro-2'-deoxypolynucleotides as templates and inhibitors for RNA- and DNA-dependent DNA polymerases.

Poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) and poly(2'-deoxycytidylic acid) (poly(dCfl)) were tested as templates in DNA synthesis reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha, mouse cell DNA polymerase gamma and avian myeloblastis virus (AMV) reverse transcriptase. Poly(dAfl).(dT)12 can fully substitute for poly(rA).(dT)12 as template with DNA polymerase gamma, to 50% with reverse transcriptase, but was poorly recognized by DNA polymerase alpha. DNA synthesis by reverse transcriptase with poly(dCfl).(dG)12 as template was 50% of that with poly(rC).(dG).(dG)12. The use of 2'-fluoropolymers as templates was more efficient at 37 degrees C than at 25 degrees C. No appreciable differences on the fidelity of DNA synthesis by reverse transcriptase were observed when dCMP misincorporation was measured with poly(dAfl).(dT)12 or poly(rA).(dT)12 as template primers. Poly(C) and poly-2'-O-methylcytidylic acid had no significant effect on the reaction catalyzed by DNA polymerase gamma and reverse transcriptase, independent of the synthetic polynucleotide complex utilized as template. On the other hand, poly(dCfl) was an inhibitor when poly(rA).(dT)12 or poly(dA).(dT)12 were used as templates, but not when poly(dAfl).(dT)12 was employed. Analogous results have been obtained with activated DNA and AMV 70 S RNA as templates in the reverse transcriptase reaction. The inhibition by poly(dCfl) was noncompetitive with regard to TTP, poly(dA) and poly(rA). Xenopus laevis oocytes DNA polymerase alpha was not inhibited by poly(dCfl).

Animals↗
PubMed1984

Stimulation of DNA synthesis by microinjection of diadenosine 5',5''-P1, P4-tetraphosphate (Ap4A) into Xenopus laevis oocytes.

Ap4A stimulated DNA synthesis when injected into oocytes. The stimulation was dramatically increased when an exogenous template was microinjected. Aphidicolin inhibited the effect of Ap4A, supporting a role of DNA polymerase alpha in this process. No stimulation by Ap4A was observed in microinjected eggs, nor ATP was able to mimic the in vivo effect of Ap4A. Besides microinjected activated DNA, the stimulation by Ap4A of DNA synthesis was also observed with poly dT and poly dT-poly dA as templates, while no effect was observed with poly dA- dT12 -18 and poly dC- dG12 -18. These results support a role of Ap4A in the initiation of DNA synthesis.

Adenine Nucleotides↗
PubMed1983

The in vitro inhibition of DNA polymerase alpha and avian reverse transcriptase by novobiocin.

Novobiocin inhibits animal DNA polymerase alpha and avian reverse transcriptase activities when these enzymes are assayed in vitro with activated DNA as template. Under the same conditions DNA polymerase beta and gamma are much less inhibited. DNA polymerase alpha and reverse transcriptase are inhibited by different mechanisms: in the case of the retroviral enzyme the effect of novobiocin is not overcome by dilution of the drug, while in the case of polymerase alpha the inhibition disappeared after novobiocin dilution. The inhibition of polymerase alpha by novobiocin is non-competitive with respect to the TTP precursor or activated DNA. The irreversible inactivation of reverse transcriptase by novobiocin leads to the loss of the enzyme affinity for primer tRNATrp. Moreover, novobiocin inhibits the partial unwinding of the 3' end of tRNATrp by reverse transcriptase.

Animals↗
PubMed1979

Multiple deoxyribonucleic acid polymerases from quiescent wheat embryos. Purification and characterization of three enzymes from the soluble cytoplasm and one from purified mitochondria.

Three DNA polymerases (A, B and C) have been purified from the soluble cytoplasm of ungerminated embryos. Mainly on the basis of chromatographic, template-specificity and salt-inhibition evidence, we have characterized the three enzymes. Other physico-chemical and enzymic properties are described. From purified mitochondria we have purified a DNA polymerase that behaves like DNA polymerase B on chromatographic and template-specificity criteria. Only highly purified enzyme B from the soluble cytoplasm showed an exonuclease activity able to degrade 3'- or 5'-labelled polydeoxyribonucleotides, as well as a 'proof-reading' capacity.

Chromatography, Ion Exchange↗
PubMed1979

DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase gamma type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called 'cytoplasmic' DNA polymerase has been found in the 10000 x g supernatant of human platelets. The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase alpha from normal cells: DEAE-cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N-ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase beta.

Blood Platelets↗
PubMed1978

[DNA polymerases in human platelets].

Human platelets have two DNA polymerases. In the mitochondrial-free cytoplasm we have found a DNA polymerase gamma and another DNA polymerase very closely related to the polymerase alpha from animal cells. In the mitochondria only the gamma activity is present.

Blood Platelets↗
PubMed1978

The inhibition of mitochondrial DNA polymerase gamma from animal cells by intercalating drugs.

DNA polymerase gamma from purified nuclei of EMT-6 cells (mice) seems to be identical to the mitochondrial DNA polymerase from the same source following several criteria. These two enzyme activities are strongly inhibited by ethidium bromide and acriflavin, while proflavin, acridine orange, daunomycin and chloroquine inhibition is less pronounced. In the case of DNA polymerases alpha and beta very little inhibition by ethidium bromide was observed. Intercalation of this dye in a poly dA-dT 12-18 template-primer was studied spectrophotometrically under conditions similar to those in the in vitro DNA polymerase assay. The polymerase assay. The inhibition by this drug of the mitochondrial DNA polymerase gamma activity was shown to be competitive at varying concentrations of TTP while the inhibition was of the non-competitive type at different concentrations of poly dA-dT 12-18. We conclude that the drug, most probably in the intercalated form, is able to interact with the active site (s) of mitochondrial DNA polymerase.

Cell Line↗
PubMed1976

Interactions of plant viral RNAs and tRNA nucleotidyl transferase.

Ribonucleic acid from TMV and BMV can accept AMP and CMP when digested with VPD. This incorporation is catalyzed by E. coli and yeast tRNA nucleotidyl transferases. Complex formation is obtained between TYMV RNA and tRNA nucleotidyl transferase in sucrose gradients while TMV and BMV RNAs failed to form a complex in the same conditions. The affinity of the enzyme for viral RNAs is lower than the affinity for tRNA as shown by complex formation on nitrocellulose filters and competition with tRNA. Coat protein from TMV particles enhances AMP and CMP incorporation onto tRNA catalyzed by the E. coli tRNA nucleotidyl transferase.

Adenosine Monophosphate↗
PubMed1973

Turnip Yellow Mosaic Virus RNA as a Substrate of the Transfer RNA Nucleotidyltransferase II. Incorporation of Cytidine 5'-Monophosphate and Determination of a Short Nucleotide Sequence at the 3' End of the RNA.

Turnip yellow mosaic virus (TYMV) RNA treated with snake venom phosphodiesterase accepts cytidine 5'-monophosphate and adenosine 5'-monophosphate (AMP) when it is incubated in the presence of cytidine 5'-triphosphate (CTP), adenosine 5'-triphosphate, and Escherichia coli transfer RNA nucleotidyltransferase; untreated TYMV RNA accepts only AMP. When alpha (32)PCTP was used for terminal labeling, the nearest neighbor analyses and the anallyses after action of various nucleases showed that the sequence of five nucleotides at the 3' end of TYMV RNA is: pGpCpApCpC. A nuclease present in commerical preparations of snake venom phosphodiesterase leads to the fragmentation of TYMV RNA, the 3' end of which is found in a fragment having a sedimentation constant close to 5s.

Journal Article↗