Showing papers by "David Baltimore published in 1974"

Potential Biohazards of Recombinant DNA Molecules

Abstract: Author(s): Paul Berg, David Baltimore, Herbert W. Boyer, Stanley N. Cohen, Ronald W. Davis, David S. Hogness, Daniel Nathans, Richard Roblin, James D. Watson, Sherman Weissman, Norton D. Zinder Source: Science, New Series, Vol. 185, No. 4148 (Jul. 26, 1974), p. 303 Published by: American Association for the Advancement of Science Stable URL: http://www.jstor.org/stable/1738673 Accessed: 20/04/2010 10:54

Is terminal deoxynucleotidyl transferase a somatic mutagen in lymphocytes

Abstract: AN enzyme able to add deoxyribonucleotides to the ends of DNA primers has been identified in calf thymus by Bollum and his colleagues and named terminal deoxynucleotidyl transferase1–4. Chang5 has indicated that the enzyme is a specialised constituent of thymus cells and is not present in any other organ of several animal species. The enzyme is located in the lymphocyte fraction of the thymus (Silverstone, Parkman, McCaffrey and Baltimore, unpublished results). Recently, we found an enzyme with the properties of a terminal transferase in circulating lymphoblasts of patients with acute lymphoblastic leukaemia (ref. 6 and McCaffrey, Harrison, and Baltimore, unpublished results).

Requirement of 3'-terminal poly(adenylic acid) for the infectivity of poliovirus RNA.

Abstract: Ribonuclease H (EC 3.1.4.34) has been used to remove selectively much of the 3'-terminal poly(adenylic acid) [poly(A)] from poliovirus RNA by treating the RNA with the enzyme in the presence of poly(dT). Over 80% of the poly(A) could be removed and the residuum was found as oligo(A) stretches attached to many or all of the viral RNA molecules. Reduction of the size of the poly(A) markedly decreased the specific infectivity of poliovirus RNA, indicating that poly(A) is necessary to the infectivity of the RNA. The virions in plaques deriving from infection with treated RNA have a normal amount and size of poly(A), indicating that mechanisms exist in infected cells to regenerate normal length poly(A) from truncated poly(A).

[66] Translation of reovirus mRNA, poliovirus RNA, and bacteriophage Qβ RNA in cell-free extracts of mammalian cells

Abstract: Publisher Summary This chapter discusses the techniques used in laboratories for the study of translation of three viral mRNA's: poliovirus RNA, reovirus mRNA, and bacteriophage Q/3 RNA. The chapter also discusses the techniques for growing the cells and the viruses and for purifying the mRNA's. The chapter has utilized two types of cell-free systems. First, preincubated extracts from several types of cultured mammalian cells. In this case incorporation of amino acids into protein is completely dependent on the addition of exogenous mRNA. Second, a non-preincubated lysate prepared from rabbit reticulocytes. In this case the addition of viral mRNA does not stimulate—and often inhibits—overall protein synthesis. However, because the predominant protein made by these lysates is globin, it is often easy to identify virus-specific proteins. The advantages of the lysate system, compared to the preincubated extracts are: (1) the amount of virus-specific protein is considerably greater, (2) the system is free of nucleases, and (3) longer completed virus polypeptides are made.

DNA polymerase activity from two temperature-sensitive mutants of Rous sarcoma virus is thermolabile.

Abstract: Temperature sensitivity of growth and transformation by certain early Rous sarcoma virus mutants correlates with thermolability of their virion-associated DNA polymerase. The enzyme is therefore encoded by viral RNA and is required for provirus synthesis.

Translation of vesicular stomatitis messenger RNA by extracts from mammalian and plant cells.

Abstract: RNA was isolated from polyribosomes of vesicular stomatitis virus (VSV)-infected cells and tested for its ability to direct protein synthesis in extracts of animal and plant cells. In cell-free, non-preincubated extracts of rabbit reticulocytes, the 28S VSV RNA stimulated synthesis of a protein the size of the vesicular stomatitis virus L protein whereas the 13 to 15S RNA directed synthesis of the VSV M, N, NS, and possibly G proteins. In wheat germ extracts, 13 to 15S RNA also directed synthesis of the N, NS, M, and possibly G proteins. Analysis of extracts labeled with formyl [35S]methionine showed that the 28S RNA directed the initiation of synthesis of one protein, whereas the 13 to 15S RNA directed initiation of at least four proteins. It is concluded that the 28S RNA encodes only the L protein, whereas the 13 to 15S RNA is a mixture of species, presumably monocistronic, which code for the four other known vesicular stomatitis virus proteins.

[13] Purification of the RNA-directed DNA polymerase from avian myeloblastosis virus and its assay with polynucleotide templates

Abstract: Publisher Summary Several enzymatic activities have been described in the purified virions of RNA tumor viruses. This chapter describes two activities that have been extensively purified: DNA polymerase that can utilize both ribo- and deoxyribonucleotide templates and ribonuclease H that selectively degrades RNA from an RNA-DNA hybrid. Several laboratories have purified DNA polymerase from avian myeloblastosis virus (AMV), Rous sarcoma virus, and Rauscher murine leukemia virus, by using a combination of ion-exchange chromatography, gel filtration, and sedimentation velocity centrifugation. The chapter describes the method of purification of AMV DNA polymerase. The enzyme is approximately 100-fold purified. Its response to various templates is discussed. The chapter concludes with a discussion on the transcription of various templates by purified AMV DNA polymerase. Purified DNA polymerase can utilize a very wide variety of polyribo and deoxyribonucleotides as templates. A primer is required to initiate DNA synthesis.

Mechanism of induction of RNA tumor viruses by halogenated pyrimidines.

Abstract: Frome these studies on JLS V-9 cells, a number of conclusions can be drawn about the mechanism of MuLV induction by halogenated pyrimidines. The compounds can induce virus from otherwise healthy cells as long as deoxycytidine is present along with the inducing agent. The compounds must be present during the S phase of the cell cycle and must be incorporated into DNA in order to induce virus (Teich et al. 1973). Only one strand of DNA need be substituted by BrdU or IdU in order to induce virus, because a one-hour period of incorporation leads to induction. From these results it is possible to construct a model for how halogenated pyrimidines are able to induce viruses from otherwise uninfected cells. Because the critical period for the incorporation of the compound is a restricted segment of the S phase of the cell, there would appear to be a critical segment of the genetic information of the cell which, when substituted with BrdU or IdU, leads to a transcriptional derepression. Presumably the critical segment of DNA is either a controlling element of the integrated provirus or it is a separate gene which controls the expression of the integrated provirus. Whichever is true, these results strongly imply that the search for specific repressors of the segments of mammalian DNA is likely to be successful and that RNA tumor viruses may offer a system in which such repression systems can be identified and investigated.

Nucleotide Sequences of Human Globin Messenger RNA

Abstract: Globin messenger RNA, isolated from human peripheral blood reticulocytes, was transcribed into complementary DNA by use of the RNA-dependent DNA polymerase of avian myeloblastosis virus. The complementary DNA was then transcribed into 32P-labeled complementary RNA by E. coli RNA polymerase in the presence of α-32P-labeled ribonucleoside triphosphates. The fingerprint pattern obtained from ribonuclease T1 digests of human globin complementary RNA was specific and reproducible. Different patterns were obtained from digests of duck, mouse, and rabbit globin complementary RNA. The fingerprint patterns obtained from digests of purified natural human 10S globin messenger RNA, labeled in vitro with 125I or with [γ-32P]ATP and polynucleotide kinase, were similar to that of the complementary RNA but contained some additional oligonucleotides. Sufficient nucleotide sequence information has been obtained from about 50% of the intermediate sized oligonucleotides (8-14 base residues long), to make possible examination of correspondence between these nucleotide sequences and globin amino-acid sequences. Approximately 70% of these oligonucleotide sequences can be matched to unique amino-acid sequences in the α- or β-globin chains. The other 30% do not match known amino-acid sequences and presumably correspond to untranslated portions of the mRNA; some of these sequences, however, can be matched to amino-acid sequence in the abnormally long segment of the α chain of hemoglobin Constant Spring, which is thought to result from a chain-termination mutation.

Hamster Leukemia Virus: Lack of Endogenous DNA Synthesis and Unique Structure of Its DNA Polymerase

Abstract: Infectious hamster leukemia virus (HaLV) contains a DNA polymerase different from those of murine and avian viruses. No endogenous reaction directed by the 60 to 70S RNA of HaLV could be demonstrated in detergenttreated HaLV virions, nor could the purified DNA polymerase copy added viral RNA. The virion RNA could, however, act as template for added avian myeloblastosis virus DNA polymerase and the HaLV DNA polymerase could efficiently utilize homopolymers as templates. The HaLV enzyme was like other reverse transcriptases in that certain ribohomopolymers were much better templates than the homologous deoxyribohomopolymers. No ribonuclease H activity could be shown in the HaLV enzyme, but neither could activity be found in the murine leukemia virus DNA polymerase. The hamster enzyme was unique in that poly(A) ·oligo(dT) was a poor template, and globin mRNA primed with oligo(dT) was totally inactive as a template. Its uniqueness was also indicated by its subunit composition; electrophoresis of the HaLV DNA polymerase in sodium dodecyl sulfate-containing polyacrylamide gels revealed equimolar amounts of two polypeptides of molecular weight 68,000 and 53,000. The sedimentation rate of the enzyme in glycerol gradients was consistent with a structure containing one each of the two polypeptides. The enzyme thus appears to be structurally distinct from other known virion DNA polymerases. Its inability to carry out an endogenous reaction in vitro might result from an inability to utilize certain primers.

Globin messenger rna in the thalassemia syndromes

Abstract: The molecular defect in thalassemia is manifest in the messenger RNA (mRNA) for globin synthesis of the thalassemic reticulocyte. Translation of thalassemic globin mRNA in heterologous cell-free protein synthesizing systems reproduces the imbalance of globin chain synthesis characteristic of the intact thalassemic reticulocyte in both aand /3-thalassemia.1-8 Studies in homozygous p-thalassemia of the Ferrara type, where there is total absence of chain synthesis (so-called Po thalassemia) , suggest that strict deficiency of functional mRNA for &chain synthesis may not be the only factor involved in that syndrome, because PA chain synthesis can apparently be induced by incubating Ferrara thalassemic polysomes in the presence of nonthalassemic supernatant fraction from Hb A or Hb S containing reticulocyte~.~+ * We have studied globin mRNA from a number of thalassemic patients of Southern Italian and Greek descent and have found, in all cases, not only deficiency of functional chain specific globin mRNA, but true quantitative deficiency of globin mRNA sequences specific for the affected globin chain in both aand 6-thalassemia.

Nucleotide sequences of human globin messenger RNA.

Abstract: The study of the nucleotide sequence of human globin messenger RNA (mRNA) has been undertaken to examine a number of questions relating to the control of globin synthesis in man. These questions concern the universality and degeneracy of the genetic code in man; the authenticity and purity of the putative 10s globin mRNA; and the primary structure of untranslated portions of globin mRNA, which may be important for mRNA stability and the control of mRNA translation, or which may be the copy of DNA sequences, adjacent to the structural gene and possibly involved in the control of mRNA synthesis. It is hoped that the study of the structure of normal human globin mRNA compared to that of mRNA obtained from various disease states may directly reveal the precise, molecular basis of certain disorders of human hemoglobin synthesis. Most techniques used in RNA sequencing rely on radioautography of :InP-

Temperature-sensitive dna polymerase from rous sarcoma virus mutants

Abstract: The DNA polymerase which can be isolated from the virions of RNA tumor viruses (the “reverse transcriptase”) has many properties which suggest that its role is to synthesize a DNA copy of the viral RNA genome (Temin and Baltimore, 1972). Only genetic experiments, however, are able to definitely establish whether it does act to copy the viral genome. Recently, Linial and Mason (1973) and Wyke (1973) have isolated and characterized temperature-sensitive mutants of Rous sarcoma virus which show temperature-sensitive synthesis of DNA in vitro. These are mutants in a function necessary only very early in the viral growth cycle. It therefore seems likely that these mutants represent temperature-sensitive DNA polymerase mutants and could be utilized to delineate the function of the DNA polymerase found in the virions of RNA tumor viruses.

Synthesis by reverse transcriptase of DNA complementary to globin messenger RNA.

Abstract: RNA tumor viruses contain a DNA polymerase that can synthesize a faithful DNA copy of viral RNA (1,5,23,25,26,31,32). This enzyme is easily released and purified from virions and can utilize a wide variety of polymers as templates (6,7,13,14,18,35,38). In order for a template to be copied, a primer or initiator that binds to the template by hydrogen bonds is required (3). The 3′-OH end of the primer is then covalently attached to the newly synthesized DNA (29). When the 60–70S tumor viral RNA is transcribed, the primer is apparently a short polyribonucleotide that is found attached to the DNA product (10,18,35,37).