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Biomedical subjects

A Panet

Publications and source records attributed to A Panet.

At least 109 records · Page 6Linked to original sources
PubMed1978

Discrimination of DNA polymerase and RNase H activities in reverse transcriptase of avian myeloblastosis virus.

The active sites in reverse transcriptase of avian myeloblastosis virus have been selectively modified by various chemical reagents. The DNA polymerase activity is very sensitive to hydrophobic sulfhydryl reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate but resistant to sulfhydryl reagents with hydrophilic properties. The RNase H activity, on the other hand, is resistant to both hydrophobic and hydrophilic sulfhydryl reagents, indicating the absence of cysteinyl residues essential for RNase H activity. N-Ethylmaleimide (NEM), an amino and sulfhydryl group specific reagent, inactivates both DNA polymerase and RNase H, the later activity being fourfold more stable. Polynucleotides, but not nucleotide triphosphates, protect the two enzymatic activites of reverse transcriptase against NEM. Since pretreatment of the enzyme with 5,5' -dithiobis(2-nitrobenzoic acid) does not prevent N-ethylmaleimide from reacting with a residue necessary for DNA polymerase activity, two different reactive groups are probably involved with this enzymatic activity. The pH profile of reverse transcriptase inhibition by N-ethylmaleimide also suggests the involvement of two reactive groups essential for the DNA polymerase activity with apparent pKas of 5.5 and 6.5. Only one reactive group with a pKa of 7.5 is found associated with the RNase H activity.

Avian Leukosis Virus↗
PubMed1978

Electron microscopic evidence for splicing of Moloney murine leukemia virus RNAs.

Poly (A) containing RNA extracted from Moloney murine leukemia virus infected mouse cells was hybridized with long single-stranded complementary DNA, prepared in detergent disrupted virions. Visualization of the hybrids in the electron microscope revealed among the structures, circles and circles with tails. Measurements performed on the circular molecules revealed two major species with circumferences corresponding to 3 and 8.2 kilobases. The latter structures had identical size to circles obtained after annealing of cDNA with the viral genome, 35S RNA. Circularization of a small viral RNA (3 kb) from infected cells in the RNA-cDNA hybrids is a direct evidence that like the 35S RNA it shares similar nucleotide sequences at both the 5' and 3' ends. The presence of 5' end sequences common to the two RNA species indicates the existence of a spliced viral RNA. Furthermore, based on the circularization of viral RNA in the hybrids, we suggest a new way to quantitate and determine the lengths of spliced RNA in retrovirus infected cells.

Base Sequence↗
PubMed1978

Binding of tRNA to reverse transcriptase of RNA tumor viruses.

The interaction of tRNA with the reverse transcriptase (RNA-dependent DNA polymerase) of mammalian RNA viruses, such as Moloney murine leukemia virus and simian sarcoma virus, has been studied. Whereas the purified reverse transcriptase of mammalian viruses sedimented in glycerol gradients as a globular protein with a molecular weight of 70,000, after interaction with tRNA the enzyme cosedimented with a protein of 150,000 molecular weight. The twofold increase in molecular weight could be a result of either two reverse transcriptase molecules complexed with a tRNA or, alternatively, several tRNA molecules bound to a single enzyme polypeptide. The enzyme complexes were dissociated in part upon degradation of the tRNA moiety by pancreatic RNase A. The reverse transcriptase released from virions of Moloney murine leukemia virus, simian sarcoma virus, and avian myeloblastosis virus, by nonionic detergent, migrated faster on glycerol gradients than purified enzyme preparation. This phenomenon was probably due to complex formation between part of the virion enzyme and the tRNA, which is endogenous in virions. Addition of exogenous tRNA was needed, however, to quantitatively complex all the virion reverse transcriptase of Moloney murine leukemia virus and simian sarcoma viruses. The reverse transcriptase of Moloney murine leukemia virus did not show tRNA species specificity in the binding reaction when glycerol gradients were used for assay. Thus, several tRNA species of Escherichia coli, yeast, chicken, and rat origin were able to complex with the enzyme. The species specificity in the interaction between tRNA and avian myeloblastosis virus reverse transcriptase was also examined. We demonstrated that under our experimental conditions, this enzyme binds different tRNA species of E. coli and yeast as well as tRNA of chicken origin.

Animals↗
PubMed1978

Binding of tryptophanyl-tRNA to the reverse transcriptase of replication-defective avian sarcoma viruses.

The ability of reverse transcriptase to bind to [3H]tryptophanyl-tRNA and to function as DNA polymerase was compared for five temperature-sensitive mutants of avian sarcoma virus. Both activities of the reverse transcriptase were found to be heat labile in LA 335 and LA 336 as compared with the wild-type parents. For the other mutant viruses, LA 338, LA 343, and LA 672, grown at the permissive temperature, the reverse transcriptase was nearly as heat stable as for the wild-type parents in terms of tRNA binding and DNA polymerase. LA 338, LA 343, and LA 672 showed characteristic defects in their reverse transcriptase when propagated at the nonpermissive temperature; namely, tryptophanyl-tRNA binding and DNA polymerase activities were coordinately decreased in these virions. The reduced enzymatic activities were not entirely due to an inactive reverse transcriptase present in the virions, however, but rather lower amounts of enzyme protein incorporated into the virions contributed to the effect, according to assays of reverse transcriptase antigen by radioimmune competition.

Animals↗
PubMed1977

Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis.

The small RNAs of Moloney murine leukemia virus (M-MuLV) were fractionated into at least 15 species by two-dimensional polyacrylamide gel electrophoresis. The pattern of small RNAs is significantly different from that of Rous sarcoma virus. A subset of the virion small RNAs is associated with the genome RNA in the 70S complex. One of the associated molecules, a cellular tRNA, is tightly bound to the genome RNA and serves as the major primer for M-MuLV RNA-directed DNA synthesis in vitro.

DNA, Viral↗
PubMed1976

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.

Base Sequence↗
PubMed1976

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction.

With the ultimate objective of the total synthesis of a tRNA gene including its transcriptional signals, an Escherichia coli tyrosine suppressor tRNA gene was chosen. The arguments in favor of this choice are presented. A plan for the total synthesis of the 126-nucleotide-long DNA duplex corresponding to a precursor (Altman S., and Smith, J. D. (1971) Nature New Biol. 233, 35) to the above tRNA is formulated. The plan involves: (a) the chemical synthesis of 26 deoxyribooligonucleotide segments, (b) polynucleotide ligase-catalyzed joining of several segments at a time to form a total of four DNA duplexes with appropriate comlementary single-stranded ends, and (c) the joining of the duplexes to form the entire DNA duplex. Ten accompanying papers describe the experimental realization of this objective.

Base Sequence↗
PubMed1976

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 8. Enzymatic joining of the chemically synthesized segments to form DNA duplexes corresponding to nucleotide sequences 23-60 and 23-66.

Polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxyribopolynucleotide segments (Fig. 1) comprising the nucleotide sequence 23-66 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been systematically investigated. Joining was studied using all possible combinations of 3, 4, and 5 and larger numbers of segments at a time. The extent of joining varied widely (0 to about 90%) in three component systems. The "self-structure" of some of the components evidently inhibited the joining. Addition of a fourth segment in general enhanced the extent of joining and optimal yields were obtained in systems containing six or more segments. A comparison of the T4-induced ligase and the E. coli polynucleotide ligase for joining of the chemically synthesized segments showed the E. coli enzyme to be inferior to the T4-induced ligase. Satisfactory syntheses of the duplexes [IIa] and [IIb] comprising, respectively, eight and seven segments were achieved in single steps. Of the two terminal segments carrying 5'-OH groups in the duplexes, only one (segment 7) was used in the prephosphorylated form. The duplexes were isolated pure and characterized by enzymatic degradations and by electrophoresis.

Base Sequence↗
PubMed1976

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 9. Enzymatic joining of chemically synthesized deoxyribopolynucleotide segments corresponding to nucleotide sequence 57-94.

The DNA duplexes representing nucleotide sequences 61-89 and 57-94 have been synthesized, isolated pure, and fully characterized. Synthesis of the duplex with the nucleotide sequence 61-89 involved the DNA ligase-catalyzed joining of chemically synthesized deoxyoligonucleotide segments 14 to 18 shown in Fig. 1A, while for the longer duplex (sequences 57-94) seven deoxyribooligonucleotides (segments 13 to 19, Fig. 1B) were used in one-step enzymatic joining. The joining of the short tetranucleotide (segment 16) to the segment 17 required the presence of the adjacent segment 14, even if the latter did not contain a 5'-phosphate group, to allow its joining to segment 16. However, in the synthesis of both of the DNA duplexes, the yields were comparatively low (30 to 40%) and could not be significantly increased although a variety of conditions was tried. The main cause in both cases evidently was the sluggish joining of segment 14 to 16 and of segment 16 to segment 17. Although the original plan for the total synthesis of this part of the gene for the tRNA precursor involved the DNA duplex consisting of segments 14 to 18, this duplex could not be quantitatively phosphorylated at the two 5'-OH ends for subsequent joining to the adjoining parts of the gene. The DNA duplex consisting of segments 13 to 19, which possesses both terminal 5'-OH groups at protruding single-stranded ends, was readily phosphorylated and used successfully in the total synthesis of the gene as described in an accompanying paper.

Base Sequence↗
PubMed1975

Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase).

The ability of tryptophan tRNA (tRNATrp) to initiate reverse transcription of the 70S RNA of avian RNA tumor viruses suggested that the reverse transcriptase (RNA-dependent DNA polymerase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) might have a specific binding site for the tRNA. A complex of tRNATrp and the avian myeloblastosis virus reverse transcriptase has been demonstrated using chromatography on Sephadex G-100 columns. Of all the chicken tRNAs, only tRNATrp and a tRNA4Met bind to the enzyme with high enough affinity to be selected from a mixture of the chicken cell tRNAs. The ability of tRNATrp to change the sedimentation rate of the enzyme indicates that tRNATrp is not binding to a contaminant in the enzyme preparation. Treatment of the enzyme with monospecific antibody to reverse transcriptase prevented binding of tRNA as well as inhibited the DNA polymerase activity of the enzyme. The ability of reverse transcriptase to utilize tRNATrp aa a primer for DNA synthesis, therefore, appears to involve a highly specific site on the enzyme.

Animals↗
PubMed1975

Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay.

A radioimmunoassay was developed that can detect and quantitate 3 ng or more of the avian RNA tumor virus reverse transcriptase. The assay detected no antigenic sites in Rous sarcoma virus alpha virions or in virions of a murine RNA tumor virus. About 70 molecules of reverse transcriptase were found per virion of avian myleloblastosis virus with this assay or with an assay based on antibody inhibition of enzymatic activity. The assay detected about 270 ng of enzyme per mg of cell protein in virus-producing cells; uninfected cells had much less antigenic material but contained some determinants able to displace radioactive antigen. No additional antigenic determinants on reverse transcriptase could be detected that were not found on the separated alpha subunit of the enzyme. Although sevenfold less sensitive than enzymatic activity as a measure of reverse transcriptase, the radioimmunoassay can detect antigen using small amounts of protein and in the presence of inhibtors.

Animals↗