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Biomedical subjects

S P Goff

Publications and source records attributed to S P Goff.

At least 19 recordsLinked to original sources
PubMed1992

Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene.

The N-myc gene encodes a putative transcription factor that is thought to function in the regulation of gene expression during cell differentiation and/or growth. To examine the role of N-myc during development, we have used targeted mutagenesis in embryonic stem cells to produce a mouse line that carries an N-myc null allele. Mice homozygous for the mutation died between 10.5 and 12.5 days of gestation. Histological analysis of mutant embryos revealed that organs and tissues expected at these stages of development were present. However, multiple defects were observed, primarily in tissues and organs that normally express N-myc. In particular, mutant hearts were underdeveloped, often retaining the S-shape more typical of 9-day-old embryos. In addition, cranial and spinal ganglia were reduced in size and/or cellularity. Most of the noted defects were more consistent with a role of N-myc in proliferation of precursor populations than with a block in differentiation per se, at least at these early stages. These results demonstrate that N-myc plays an essential role during development and clearly confirm that N-myc has a physiological function that is distinct from that of the other myc-family genes.

Alleles↗
PubMed1992

Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo.

Animals↗
PubMed1992

Mutagenesis of the Glu-89 residue in human immunodeficiency virus type 1 (HIV-1) and HIV-2 reverse transcriptases: effects on nucleoside analog resistance.

A Glu-89-->Gly alteration in the human immunodeficiency virus type 1 reverse transcriptase (RT) was previously shown to result in resistance to several dideoxynucleoside analogs and to phosphonoformic acid (PFA; foscarnet). This residue was altered to Ala, Val, Ser, Thr, Gln, Asp, Asn, or Lys, and the ddGTP and PFA sensitivities of the mutant RTs were measured. Replacements with Ala, Gly, Val, and Thr led to resistance to inhibition by ddGTP, while mutants with amino acid Ser, Gln, Asn, Asp, or Lys displayed only moderate or no resistance. A similar result was obtained with inhibition by PFA, except that the Asp-89 mutant also displayed resistance. Furthermore, the introduction of Glu-89-->Gly alteration into the RT of human immunodeficiency virus type 2 likewise rendered it resistant to both ddGTP and PFA.

Amino Acid Sequence↗
PubMed1992

Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H.

We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT. This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H. This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay. However, proviruses reconstructed to include this deletion were noninfectious. Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT. The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.

3T3 Cells↗
PubMed1992

A mutation at one end of Moloney murine leukemia virus DNA blocks cleavage of both ends by the viral integrase in vivo.

The integration of retroviral DNA proceeds through two steps: trimming of the termini to expose new 3' OH ends, and the transfer of those ends to the phosphates of target DNA. We have examined the ability of the Moloney murine leukemia virus integrase protein (IN) to trim the termini of the preintegrative DNA of mutant viruses with alterations in the U3 inverted repeat. The mutant terminus of one replication-defective viral DNA, containing a 7-bp deletion in the U3 inverted repeat, was not trimmed to produce the normal recessed end. Remarkably, the other terminus of this mutant DNA was also not trimmed, even though its sequence is wild type. This finding suggests that the IN protein requires the presence of two good ends before becoming properly activated to trim either one.

Base Sequence↗
PubMed1992

Genetic assay for multimerization of retroviral gag polyproteins.

We have established a genetic assay for the multimerization of retroviral gag polyproteins. This assay is based on the GAL4 two-hybrid system for studying protein-protein interactions (S. Fields and O. Song, Nature (London) 340:245-246, 1989). In our initial experiments, we generated Saccharomyces cerevisiae plasmids that separately express the GAL4 DNA-binding and GAL4 activation domains fused to the human immunodeficiency virus type 1 (HIV-1) gag polyprotein, Pr55gag. The coexpression of these two hybrid proteins in S. cerevisiae results in the association of the GAL4 domains and the potent activation of an integrated GAL4-responsive lacZ indicator gene. Similar results were obtained with plasmids encoding GAL4-Moloney murine leukemia virus (M-MuLV) gag polyprotein hybrid proteins. In contrast, the heterologous GAL4-HIV-1 gag and GAL4-M-MuLV gag fusion proteins were unable to interact with each other to induce lacZ expression. The results suggest that this yeast system provides a rapid and specific assay for the interactions of retroviral gag proteins that occur during virion assembly.

Cloning, Molecular↗
PubMed1992

Genetics of retroviral integration.

Today the retroviral integration reaction is probably understood, both in terms of its genetics and chemistry, in as much detail as any eukaryotic recombination process. That understanding is in part due to its high efficiency (for it can be induced to occur synchronously in every cell of a culture); to its simplicity (for there is only one major protein player); to its accessibility (for the viral genome has provided all the cis- and trans-acting players); and to its willingness to perform well in vitro, ultimately with purified components. The process has thus made the classic transition from a phenomenon to be studied genetically to a reaction that can also be studied biochemically. The next advances in our understanding of the process of retroviral integration are likely to center on chemical issues. Some basic enzymological issues need to be addressed: we need to determine the oligomeric state of the native IN protein; its state when bound to linear viral DNA; the residues at the active site; the residues involved in sequence-specific recognition of DNA; and the points of contact between IN monomers. Much of this information will follow from detailed mutagenesis of expressed IN genes. A crucial step will be the determination of the structure of the IN protein at atomic resolution through X-ray diffraction analysis of protein crystals, a project underway in several laboratories. That structure may immediately suggest how the enzyme contacts and joins two DNA molecules, and will enormously facilitate the design and interpretation of mutational studies. It seems plausible that we can understand the IN protein as a machine as well as any nuclease or recombinase. A significant number of larger biological questions about integration remain unanswered and will require genetic approaches. What is the true structure of the preintegration complex in the cytoplasm? How does the complex enter the nucleus, and obtain access to the host DNA? Why, at least for most viruses in most cells, does integration depend on cell division? Why does efficient expression of the viral DNA to form progeny viral RNA and proteins depend on integration? How are target sites for integration on the host genome selected, and why are there "hot spots" for insertion? Are there host proteins that facilitate or participate in the integration reaction itself, and what are those proteins? Are any of those proteins involved in site selection?(ABSTRACT TRUNCATED AT 400 WORDS)

DNA Mutational Analysis↗
PubMed1992

Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities.

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

Amino Acid Sequence↗
PubMed1992

Bone marrow-transforming activity of linker insertion mutants of Abelson murine leukemia virus.

Two sets of mutants of the Abelson murine leukemia virus, generated by linker insertion mutagenesis of a cloned proviral DNA, were tested for their ability to transform bone marrow cultures in vitro. All the viruses retained an intact tyrosine kinase domain and were competent for transformation of NIH3T3 fibroblasts in culture. One series contained 12-bp linker insertions in the regions flanking the kinase domain, and the other contained frameshift mutations that truncated the gene product downstream of the kinase domain. The majority of the 12-bp insertion mutants retained full bone marrow-transforming activity; only one insertion in the SH2 domain showed reduced activity. This mutant suggests that some aspect of the SH2 domain may be more important in transformation of lymphocytes than fibroblasts. In contrast to the first set of mutants, the bone marrow-transforming activity of the majority of the truncation mutants was significantly reduced or completely lost. We conclude that there is a broad requirement for an intact C-terminal domain of the v-abl protein for the transformation of pre-B cells, but that no single part of this domain is critical.

3T3 Cells↗
PubMed1991

Isolation and characterization of a dideoxyguanosine triphosphate-resistant mutant of human immunodeficiency virus reverse transcriptase.

The appearance of drug-resistant strains of viral pathogens is a major difficulty confounding current efforts to block viral infections. The identification and analysis of mutations responsible for drug resistance can provide important clues helpful in understanding the mechanisms of resistance and in the eventual development of better therapies. We have used a direct screening method to scan libraries of mutagenized genes encoding the reverse transcriptase of human immunodeficiency virus type 1, and have recovered a variant enzyme that is resistant to the chain-terminator inhibitor 2',3'-dideoxyguanosine triphosphate. The single substitution mutation in this variant conferred broad crossresistance to a variety of other antiviral compounds currently in clinical trials. Virus carrying the mutation was fully infectious in cultured human lymphocytes. The replication of the mutant virus was highly resistant to phosphonoformic acid but did not show increased resistance to the prodrug dideoxyguanosine.

Antiviral Agents↗
PubMed1991

Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations.

The c-abl gene, originally identified as the cellular homolog of the transforming gene of the Abelson murine leukemia virus, encodes a protein-tyrosine kinase of unknown function that is expressed in all mammalian tissues. We have previously described the introduction of a mutation in the c-abl gene into the mouse germline via targeted gene disruption of embryonic stem cells. We now show that mice homozygous for this mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. We have examined components of the immune system and have found major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments.

Animals↗
PubMed1991

Analysis of mutations in the envelope gene of Moloney murine leukemia virus: separation of infectivity from superinfection resistance.

Six deletion mutations and an insertion were generated in the env gene of cloned copies of Moloney murine leukemia virus DNA. All seven mutants were replication-defective as tested by transformation of NIH/3T3 cells. The mutant DNAs were introduced into NIH/3T3 cells to generate stable producer lines; all released virion particles into the medium, suggesting that none of the mutations affected overall viral gene expression, gag and pol gene expression, gag and pol gene functions, or virion budding. Several of the mutations reduced the lifetime of the env protein or blocked its export to the cell surface. One mutation altering the membrane-spanning region and the cytoplasmic tail of the TM protein had no effect on export of the protein, proteolytic processing, or incorporation into virion particles, but still blocked the infectivity of the resulting virus. The results suggest that alterations in the transmembrane region can affect early steps of infection, such as the fusion of virion and host membranes. Cells expressing this mutant env protein were fully resistant to superinfection by wild-type virus. Thus, induction of virus resistance, presumably reflecting blocking the virus receptor, can be separated from virus infectivity.

Amino Acid Sequence↗
PubMed1991

Generation of recombinant murine retroviral genomes containing the v-src oncogene: isolation of a virus inducing hemangiosarcomas in the brain.

A series of recombinant retroviral genomes was generated by cotransformation of NIH 3T3 cells with a mixture of cloned DNAs: a proviral copy of the wild-type Moloney murine leukemia virus, and Moloney-based vectors containing defective copies of the chicken v-src and the murine v-abl oncogenes. Morphologically transformed foci, appearing at low frequencies in these cultures, released high titers of transforming viruses. Analysis of one group of these viruses showed that the genomes were recombinants containing portions of the viral gag gene juxtaposed to the v-src oncogene. Biologically active cloned DNAs of two of these viruses were obtained and mapped in detail. One of these viruses did not cause disease after inoculation into newborn mice, but the other induced rapidly fatal hemangiosarcomas located exclusively in the brain.

Amino Acid Sequence↗
PubMed1991

Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.

Base Sequence↗
PubMed1991

Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function.

The reverse transcriptase enzymes of retroviruses are multifunctional proteins containing both DNA polymerase activity and a nuclease activity, termed RNase H, specific for RNA in RNA-DNA hybrid form. To determine the role of RNase H activity in retroviral replication, we constructed a series of mutant genomes of Moloney murine leukemia virus that encoded reverse transcriptase enzymes that were specifically altered to retain polymerase function but lack RNase H activity. The mutant genomes were all replication defective. Analysis of in vitro reverse transcription reactions carried out by mutant virions showed that minus-strand strong-stop DNA was formed but did not efficiently translocate to the 3' end of the genome; rather, the DNA was stably retained in RNA-DNA hybrid form. Plus-strand strong-stop DNA was not detected. These results suggest that RNase H normally promotes strong-stop translocation, perhaps by exposing single-stranded DNA sequences for base pairing. Four new DNA species were also detected among the reaction products. Analysis of these DNAs suggested that they were minus-strand DNAs formed from VL30 RNAs encoded by the mouse genome. We suggest that reverse transcriptase can initiate DNA synthesis at any one of four alternate tRNA primer-binding sites near the 5' ends of VL30 RNAs.

Animals↗
PubMed1990

Rapid reversion of a deletion mutation in Moloney murine leukemia virus by recombination with a closely related endogenous provirus.

During abortive infection of mouse cells, defective retroviruses carrying deletions in essential functions can recombine with endogenous retroviral sequences to form viable, replication-competent viruses. We have examined the reversion of a mutant Moloney murine leukemia virus with a deletion in the protease domain of the pol gene after infection of NIH/3T3 cells. In this system revertants arise quickly, only 2 weeks after infection. Analysis of DNA clones of the revertant viral genomes showed that they were derived by recombination with a long sequence of gag and pol exhibiting 95% sequence identity to Moloney virus. One such cloned recombinant was fully infectious, indicating that the repertoire of viral sequences in the NIH/3T3 genome must include substantial stretches of functional viral genes. Examination of the viral DNAs very early in the infection revealed the presence of defective genomes, formed by nonhomologous crossovers between the two parental sequences. We suggest that these may serve as intermediates in the eventual formation of the viable revertant genomes.

Amino Acid Sequence↗
PubMed1990

Expression of enzymatically active reverse transcriptase of simian immunodeficiency virus in bacteria: sensitivity to nucleotide analogue inhibitors.

A fragment of the SIVmac251 pol gene was expressed in Escherichia coli as a trpE fusion protein. Analysis of extracts from bacteria containing this expression plasmid revealed the presence of a reverse transcriptase activity dependent on Mg2+ as divalent cation and active on both poly(rA).oligo(dT) and poly(rC.oligo(dG) templates. In comparative studies, the SIV and HIV-1 reverse transcriptases expressed in bacteria displayed very similar high sensitivities to the chain terminator inhibitors AZTTP and ddTTP. The reverse transcriptase of Moloney murine leukemia virus and the DNA polymerase of E. coli were both more resistant to ddTTP, and the E. coli enzyme was significantly more resistant to AZTTP.

Dideoxynucleotides↗