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Biomedical subjects

L A Donehower

Publications and source records attributed to L A Donehower.

81 records · Page 5Linked to original sources
PubMed1988

Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3' region of pol.

Twelve linker insertion mutations have been constructed in the 3' part of the pol gene of Moloney murine leukemia virus. This region of the Moloney murine leukemia virus genome encodes IN or p46pol, which is required for integration of the retroviral DNA into the host cell chromosome. Viral proteins synthesized by these mutants were used to pseudotype a neo-containing retroviral vector. Ten of twelve linker insertion mutant pseudotypes were unable to generate stable proviruses in infected mouse cells, as measured by the formation of G418-resistant colonies. Two mutants mapping at the 3' terminus of the IN-encoding region were competent for the formation of stable vector proviruses (hundreds of G418-resistant colonies per mutant pseudotype-infected plate). Representative linker insertion mutants were also tested for the ability to synthesize viral unintegrated DNA in newly infected cells. All assayed mutants were capable of synthesizing all normal forms of viral unintegrated DNA. The structure of integrated vector proviruses generated by defective and nondefective linker insertion mutants was also analyzed. All replication-competent mutants generated normal proviruses, while the few obtainable proviruses generated by replication-defective mutants were sometimes aberrant in structure. These results argue strongly (and confirm previous data) that the IN-encoding region of pol does not play a significant role in DNA synthesis, but is absolutely required for the formation of normal proviral DNA.

Animals↗
PubMed1988

Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma.

Hepatitis B virus (HBV) is clearly a factor in the development of hepatocellular carcinoma, but its mechanism of action remains obscure. One possibility is that the HBV integration event alters the expression of a nearby growth-regulatory cellular gene. A 9-kilobase (kb) DNA fragment containing an HBV insert plus flanking cellular sequences was cloned from a hepatoma specimen from Shanghai, People's Republic of China. Restriction mapping of the insert revealed a large inverted repeat structure consisting of both viral sequences (encompassing all of the core and pre-S regions and portions of the X and S genes) and at least 3 kb of unique cellular sequences. The virus-cell junction mapped 11 nucleotides from the DR1 region, in a position within the HBV X gene and included in the cohesive overlap region. A probe generated from 1.0 kb of the flanking cellular DNA mapped the viral insert to chromosome 17 in the region designated 17p11.2-17p12, which is near the human proto-oncogene p53. Sequence data from a portion of the flanking cellular DNA revealed a stretch of approximately 70 base pairs that showed highly significant homology with a conserved region of a number of functional mammalian DNAs, including the human autonomously replicating sequence 1 (ARS1).

Base Sequence↗
PubMed1987

Retroviral DNA integrated during infection by an integration-deficient mutant of murine leukemia virus is oligomeric.

Mutants with lesions engineered into the 3' region of the pol gene of murine leukemia virus (MLV), the region that encodes p40pol, have allowed us to define a function that is required for the normal integration of retroviral DNA. Two such mutants, MLV-SF1, which bears a single missense mutation (L. A. Donehower and H. E. Varmus, Proc. Natl. Acad. Sci. USA 81:6461-6465, 1984), and MLV-SF2, which has a frameshift mutation at the same site, have been used to explore the consequences of integration deficiencies. Pseudotypes that were formed with viral proteins supplied by these mutants and a replication-defective genome that carried a selectable genetic marker induced colony formation at least 100-fold less efficiently than did pseudotypes that were formed with proteins encoded by wild-type helper virus. Most of the proviruses from the mutant pseudotypes were manifestly aberrant when they were analyzed by mapping with restriction enzymes. Rare proviruses that appeared to be normal by restriction enzyme mapping were molecularly cloned and subjected to more detailed study. Two proviruses resulting from infection with the MLV-SF1 pseudotype were identical or nearly identical to wild-type MI V proviruses. Two base pairs were missing from the ends of the long terminal repeats at the host-viral junctions, and 4- or 5-base-pair duplications of host DNA flanked the proviruses. Thus, the missense mutant appears to retain a low level of normal integration activity. Four proviruses from cells that were infected with the MLV-SF2 pseudotype were flanked on both sides by viral DNA of either helper or vector origin, with a single long terminal repeat at each end of the intact vector DNA and with irregular junctions of viral and host DNAs. These results are most simply explained by the nonspecific integration of dimeric or trimeric forms of viral DNA, which may arise during infection by replicative or recombinational mechanisms.

Animals↗
PubMed1984

A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration.

We have used site-directed mutagenesis of cloned Moloney murine leukemia virus (MuLV) DNA to define a function encoded in the 3' region of the viral pol gene and required for efficient integration of viral DNA. One mutant, MuLV-SF1, contained a single base substitution (C to T at base 4950) that resulted in an arginine to cysteine change in a region highly conserved among retroviruses. Mutant DNA, introduced into rat cells by cotransfection with a herpes simplex virus thymidine kinase gene (HSV tk), directed production of virus particles with reverse transcriptase activity. Infection of cells with these particles led to synthesis of full-length linear and circular forms of unintegrated viral DNA; however, integrated viral DNA was decreased at least by a factor of 10 when examined by DNA hybridization, and the mutant particles were less efficient then wild-type virus at establishing an infection by a factor of at least 300. Pseudotypes formed with the proteins of MuLV-SF1 and the genome of a replication defective marker MuLV, carrying the HSV tk gene, were less effective by at least a factor of 100 in producing tk+ colonies than pseudotypes formed with proteins encoded by wild-type virus. When the MuLV-SF1 pseudotypes did produce tk+ cells, most of the proviruses were integrated aberrantly. We conclude that the MuLV-SF1 pol gene is defective for a function that is required for normal integrative recombination and dissociable from DNA synthesis.

Cell Transformation, Viral↗
PubMed1983

Further evidence for the protein coding potential of the mouse mammary tumor virus long terminal repeat: nucleotide sequence of an endogenous proviral long terminal repeat.

The 3' half of an endogenous mouse mammary tumor virus from a C3H mouse was cloned in the Charon 4A vector phage. A comparison of the proviral clone with previously published endogenous mouse mammary tumor virus restriction maps identified it as endogenous unit II (J. Cohen and H. Varmus, Nature [London] 278:418-423, 1979), which is present in all inbred mouse strains derived from the original Bagg albino x DBA cross. The nucleotide sequence of the unit II long terminal redundancy (LTR) was determined and compared with the sequence previously determined for the exogenous C3H virus LTR (Donehower et al., J. Virol. 37:226-238, 1981). Virtually all sequence differences between the two LTRs were base substitutions. The total amount of sequence divergence was 6.6%. The large open reading frame reported previously in the exogenous LTR was preserved in the endogenous LTR. In addition, the pattern of sequence divergence was highly nonrandom with respect to the putative amino acid codons of the two open reading frames. Most of the base substitutions in this region resulted in silent or conservative amino acid codon changes. The nonrandom divergence pattern indicates that selective forces are operating on this segment of DNA and argues that the putative protein is functional in the life cycle of mouse mammary tumor virus. Possible roles for the protein and its mode of expression are discussed.

Amino Acid Sequence↗
PubMed1981

Regulatory and coding potential of the mouse mammary tumor virus long terminal redundancy.

Molecular clones containing the 3' half of newly integrated mouse mammary tumor virus (MMTV) DNA with adjacent mouse cellular sequences were characterized. In addition, we cloned the long terminal redundancy joint from the unintegrated circular form of MMTV DNA. The entire nucleotide sequence of the integrated and part of the unintegrated terminal redundancy was determined; this allowed us to delineate the boundaries of the MMTV long terminal redundancy, which comprises 1,327 base pairs. The position of possible RNA polymerase II initiation and termination signals corresponded closely to the expected regions of viral RNA initiation and termination specified by current models. The MMTV long terminal redundancy also contained a large open reading frame with sufficient information for a protein of 198 amino acids. Initial comparison of flanking 3' cellular sequences from three independent integrated clones suggested there was no host sequence specificity in the MMTV integration event. However, specificity of integration with respect to viral sequences was precise.

Bacteriophage lambda↗
PubMedDate not supplied

Cancer prevention studies in p53-deficient mice.

Future progress in mechanism-based cancer prevention research may be facilitated by animal models displaying specific genetic susceptibilities for cancer, such as mice deficient in 1 (+/-) or both (-/-) alleles of the p53 tumor suppressor gene. We observed in p53-/- mice that calorie restriction (CR) increased the latency of spontaneous tumor development (mostly lymphomas) by approximately 75%, decreased serum insulin-like growth factor-1 (IGF-1) and leptin levels, slowed thymocyte cell cycle traverse, and induced apoptosis in immature thymocytes. In p53+/- mice, CR and a 1 d/wk fast each delayed spontaneous tumor development (a mix of lymphomas, sarcomas, and epithelial tumors) and decreased serum IGF-1 and leptin levels, even when begun late in life. In p53+/-Wnt-1 transgenic mice, a mammary tumor model, the same interventions increased mammary tumor latency and reduced mean serum IGF-1 and leptin levels to <50% of those of control mice. We capitalized on the susceptibility of p53+/- mice to chronic, low-dose aromatic amine-induced bladder carcinogenesis to develop a useful model for evaluating bladder cancer prevention approaches. These examples clearly indicate that mice with specific (and humanlike) genetic susceptibilities for cancer are powerful models for testing interventions that may inhibit carcinogenesis in humans.

Animals↗