Showing papers by "David Baltimore published in 1975"

Terminal deoxynucleotidyl transferase activity in human leukemic cells and in normal human thymocytes.

Abstract: Peripheral leukocytes from patients with and without leukemia were assayed for presence of terminal deoxynucleotidyl transferase. Activity of this enzyme was detected in circulating leukemic cells from 11 to 13 patients with acute lymphoblastic leukemia, and in one of four with chronic myelogenous leukemia in blast crisis, but not in leukocytes from patients with other kinds of leukemia or in normal leukocytes. Its presence in a patient with chronic myelogenous leukemia in blast crisis lends biochemical support to the suggestion that some patients with chronic myelogenous leukemia undergo a lymphoblastic rather than a myeloblastic crisis. The thymocyte and leukemic-cell enzyme have the same substrate and primer preference. Normal thymocytes and leukemic cells contain two forms of terminal deoxynucleotidyl transferase that can be separated by phosphocellulose chromatography. The enzyme may provide a means for classifying leukemic cells on a biochemical basis independently of classic morphologic and clinical criteria.

Summary statement of the Asilomar conference on recombinant DNA molecules.

Abstract: This meeting was organized to review scientific progress in research on recombinant DNA molecules and to discuss appropriate ways to deal with the potential biohazards of this work. Impressive scientific achievements have already been made in this field and these techniques have a remarkable potential for furthering our understanding of fundamental biochemical processes in pro- and eukaryotic cells. The use of recombinant DNA methodology promises to revolutionize the practice of molecular biology. Although there has as yet been no practical application of the new techniques, there is every reason to believe that they will have significant practical utility in the future.

In vitro transformation of lymphoid cells by Abelson murine leukemia virus

Abstract: Cell cultures prepared from fetal murine liver were infected by Abelson murine leukemia virus After about 2 weeks, proliferating cells of lymphoid morphology appeared in some of the cultures Addition of 2-mercaptoethanol to the initial culture medium greatly enhanced the appearance of the lymphoid cells Immunoglobulin determinants were evident on the cells in some cultures Continuous passage of the cells in certain cultures was possible and the passaged cells could form tumors after animal inoculation Because Abelson murine leukemia virus is able to induce in vitro malignant transformation of lymphoid cells, it probably causes leukemia by directly affecting cellular growth control

Murine terminal deoxynucleotidyl transferase: cellular distribution and response to cortisone.

Abstract: The mouse thymus contains two forms of terminal deoxynucleotidyl transferase (TdT) which are distinguishable by the salt concentration necessary to elute them from a phosphocellulose column, by their distrubtion among the thymocyte subpopulations, and by their sensitivity to cortisone treatment. In the whole thymus the later eluting peak (peak II) is the predominant one with about 3-10% of the total activity appearing in peak I. Both peak I and peak II activities are most sensitively assayed by the polymerization of dGMP onto an oligo(dA) primer. The minor population of thymocytes which is less dense and cortisone-resistant contains a higher specific activity of peak I TdT. The majority of TdT activity is, however, found in the major population of thymocytes which occurs in the center region of a bovine serum albumin gradient and is cortisone-sensitive. A very low level of an activity indistinguishable from peak II TdT activity is also detected in the mouse bone marrow. Other tissues, such as spleen, liver, heart, and brain lack detectable amounts of TdT activity.

Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay.

Abstract: 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.

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

Abstract: 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 2777) 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

Complete translation of poliovirus RNA in a eukaryotic cell-free system

Abstract: Poliovirus RNA stimulates imcorporation of 35S from both [35S]methionine and formyl-[35S]methionyl-tRNAfMet in cell-free systems derived from HeLa cells or from poliovirus-infected HeLa cells. The largest product formed under the direction of the viral RNA is the same size as the polyprotein thought to represent translation of the entire RNA. Synthesis of this polyprotein and other large products was stimulated greatly by increasing the salt concentration during the reaction from the optimum for initiation (90 mM) to the optimum for elongation (155 mM). Only one initiation peptide could be identified, and a tryptic digest of the product contained mainly peptides that cochromatographed with peptides from authentic viral proteins. The RNA from a deletion mutant of poliovirus initiated protein synthesis at the same site used by standard RNA and programmed synthesis of an appropriately deleted set of polypeptides. The results strongly support the model of translation of poliovirus RNA from a single initiation site into a continuous polyprotein that is cleaved to form the functional proteins. It is suggested that uninfected HeLa cell extracts can carry out the cleavages of nascent polyprotein.

Polyadenylic acid on poliovirus RNA. II. poly(A) on intracellular RNAs.

Abstract: The content, size, and mechanism of synthesis of 3'-terminal poly(A) on the various intracellular species of poliovirus RNA have been examined. All viral RNA species bound to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. At 3 h after infection, the poly(A) on virion RNA, relicative intermediate RNA, polyribosomal RNA, and total cytoplasmic 35S RNA was heterogeneous in size with an average length of 75 nucleotides. By 6 h after infection many of the intracellular RNA's had poly(A) of over 150 nucleotides in length, but the poly(A) in virion RNA did not increase in size suggesting that the amount of poly(A) which can be encapsidated is limited. At all times, the double-stranded poliovirus RNA molecules had poly(A) of 150 to 200 nucleotides. Investigation of the kinetics of poly(A) appearance in the replicative intermediate and in finished 35S molecules indicated that poly(A) is the last portion of the 35S RNA to be synthesized; no nascent poly(A) could be detected in the replicative intermediate. Although this result indicates that poliovirus RNA is synthesized 5' leads to 3' like other RNA's, it also suggests that much of the poly(A) found in the replicative intermediate is an artifact possibly arising from the binding of finished 35S RNA molecules to the replicative intermediate during extraction. The addition of poly(A) to 35S RNA molecules was not sensitive to guanidene.

Effect of the Fv-1 locus on the titration of murine leukemia viruses.

Abstract: Titration of N- and B-tropic murine leukemia viruses on sensitive and resistant cell lines has been studied by direct XC plaque assay and infective center assay. The titration of cloned B-tropic virus by infective center assay on BALB/3T3 (Fv-1b/b) and NIH/3T3 (Fv-1n/n) cells gave one-hit patterns, with 100-fold less infected NIH/3T3 cells than BALB/3T3 cells. The titration of B-tropic virus on DBA/2 cells (Fv-1n/n) was also a one-hit. The titration of a one-hit curve, and there were about 100-fold less infected BALB/3T3 cells than NIH/3T3 cells. Comparable results were obtained by titrating the cloned N-tropic virus on congenic SIM (Fv-1n/n) and SIM.R (Fv-1b/b) cells or the Gross N-tropic virus on BALB/3T3 cells. Therefore, our data indicate that the multiple-hit phenomenon described previously may not be an essential part of the Fv-1 gene restriction.

Screening procedure for complementation-dependent mutants of vesicular stomatitis virus.

Abstract: To isolate new types of vesicular stomatitis virus (VSV) mutants, a four-stage screen was developed which identifies and characterizes mutants capable of complementing the defect in the VSV temperature-sensitive mutant tsG11. Two types of mutants of VSV, Indiana serotype, have been found by using the screen; they are new temperature-sensitive mutants which are, of necessity, not in complementation group I and mutants which do not produce plaques under conditions of single infection at 31 C (the normal permissive temperature) and are, therefore, called complementation-dependent mutants. The newly isolated, temperature-sensitive mutants fall into three complementation groups, two of which are congruent with known complementation groups; the newly identified group extends to six the number of complementation groups of VSV Indiana. The nature of the complementation-dependent mutants has not been established, but one was shown to not contain a significant deletion in its nucleic acid.

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

Abstract: A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts

Poly(A) on Mengovirus RNA

Abstract: The content and size of the poly(A) on Mengovirus RNA grown in both mouse L cells and HeLa cells have been examined. Virion RNA from either cell line could bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. The size of the poly(A) on the Mengovirus RNA was independent of the host cell and averaged from 50 to 70 nucleotides.

The molecular biology of poliovirus.

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Use of Pseudotypes of Vesicular Stomatitis Virus to Study Cellular Resistance to Murine Rna Tumor Viruses

Abstract: Pseudotypes of vesicular stomatitis virus (VSV) genomes coated by the surface envelope from an N-tropic or a B-tropic tumor virus grow equally well in cells homozygous for either the Fv-1 n or Fv-1 p alleles Therefore, the product of the Fv-1 locus, which restricts growth of murine RNA tumor viruses, must act on an intracellular aspect of tumor virus replication, a step following attachment and penetration