Structure of cloned retroviral circular DNAs: implications for virus integration.
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Biomedical subjects
M Paskind
Publications and source records attributed to M Paskind.
Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: implications for retrovirus integration.
Closed circular Moloney murine leukemia virus (M-MuLV) DNA was prepared from recently infected cells and cloned in a lambda vector. Four classes of cloned M-MuLV inserts were found: Class I, full length 8.8-kilobase (kb) inserts with two tandem long terminal repeats (LTRs) of 600 base pairs; class 2, 8.2-kb inserts with a single copy of a LTR; class 3, M-MuLV DNA inserts with various portions deleted; and class 4, an 8.8-kb insert with an internal sequence inversion. Determination of nucleotide sequence at the junction between the two LTRs from a class 1 insert suggested that circularization occurred by blunt-end ligation of an 8.8-kb linear DNA. The class 4 molecule had an inversion that was flanked by inverted LTRs, each of which had lost two terminal base pairs at the inversion end points. Also, four base pairs that were present only once in standard M-MuLV DNA were duplicated at either end of the inversion. This molecule was interpreted as resulting from an integrative inversion in which M-MuLV DNA has integrated into itself. Its analysis thus provided explicit information concerning the mechanism by which retrovirus DNA integrates into host cell DNA. Models of retrovirus integration based on bacterial DNA transposition mechanisms are proposed.
Bacteriophage lambda↗
Structure of the Abelson murine leukemia virus genome.
Virions produced from cells transformed by A-MuLV contain a 30S, 5.6 kb RNA that can be translated in a cell-free system to form the characteristic A-MuLV protein. This RNA was mapped by heteroduplex methods using DNA probes from M-MuLV, the presumed parent of A-MuLV. The overall organization of the RNA was determined by using full-length M-MuLV reverse transcribed DNA and visualizing the heteroduplexes in the electron microscope. This showed that A-MuLV and M-MuLV have homologous sequences at both ends of their RNAs but that the central portion of the A-MuLV genome is not homologous to sequences in M-MuLV RNA. A precise measure of the lengths of the shared regions was obtained by using S1 nuclease to digest hybrids between 32P-labeled M-MuLV DNA and A-MuLV RNA; the resulting fragments were analyzed for their length by electrophoresis. The regions of homology were shown to be 1320 nucleotides long at the 5' end and 730 nucleotides long at the 3' end. Thus approximately 6200 nucleotides of the approximately 8300 in M-MuLV RNA were deleted when the A-MuLV genome was formed, but an insert of 3600 nucleotides, presumably derived from the normal murine genome, was inserted in place of the deleted region.
Abelson murine leukemia virus↗
Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome.
A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus.
Animals↗
Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblast and lymphoid cells.
Extracts from lymphoid and fibroblast cell lines transformed by Abelson murine leukemia virus (A-MuLV) contain a protein of molecular weight 120,000 (P120). Immunoprecipitation with specific sera shows that P120 contains regions homologous to the 5'-terminal segment of the MULV gag gene complex--p15, p12, and at least part of p30--but lacks detectable determinants of p10, reverse transcriptase, and the envelope glycoprotein. P120 is phosphorylated and has an intracellular half-life of 3--6 hr. In vitro translation of virion RNA from A-MuLV, with Moloney MuLV as helper, yields a product of molecular weight 120,000 with serological reactivity similar to that of the cellular P120. Translation of the RNA from the helper gave no P120. P120 is expressed in all lymphoid and fibroblastic cell lines we have tested that were transformed by A-MuLV but is not detectable in a lymphoid line in which the A-MuLV genome was established by infection but was not responsible for the transformation. Expression of P120 is selectively retained in clones of A-MuLV-transformed lymphocytes that convert to a nonproducer state after loss of expression of helper MuLV intracellular precursors. These results suggest that the P120 product of the A-MuLV genome may be responsible for maintenance of the transformed phenotype of lymphoid and fibroblast cells transformed by the virus.
Animals↗
Mechanism of induction of RNA tumor viruses by halogenated pyrimidines.
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.
Bromodeoxyuridine↗
Measurement of the sequence complexity of cloned Moloney murine leukemia virus 60 to 70S RNA: evidence for a haploid genome.
The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.
Animals↗
Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines.
The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire of VH genes expressed in pre-B cells is strikingly different from that seen in mature populations.
Amino Acid Sequence↗
Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells.
The variable regions of immunoglobulin heavy chains are encoded in the germ line by three discrete DNA segments: VH (variable) elements, D (diversity) elements and JH (joining) elements. During the differentiation of B lymphocytes, individual segments from each group are brought together by recombination to form the complete VHDJH variable region. To understand these processes better, we have now isolated and sequenced molecular clones representing intermediates (DJH fusions) and final products (VH-to-DJH joins) of heavy-chain gene rearrangement in two cell lines that represent analogues of cells at early stages of B-lymphocyte differentiation. Heavy-chain gene assembly in one cell line but not in the other is accompanied by the appearance of short nucleotide insertions at the recombinational junctions. The generation of such insertions is positively correlated with the expression of terminal deoxynucleotidyl transferase in these lines.
Animals↗