Sexual transmission of an HIV-1 variant resistant to multiple reverse-transcriptase and protease inhibitors.
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Anti-HIV Agents↗
Biomedical subjects
C J Petropoulos
Publications and source records attributed to C J Petropoulos.
Advances in quantitative PCR technology: 5' nuclease assays.
The development of 5' nuclease assays represents a significant advance in nucleic acid quantitation. This approach utilizes the 5'-3' exonuclease activity of Thermus aquaticus (Taq) polymerase to cleave a dual-labelled probe annealed to a target sequence during amplification. The release of a fluorogenic tag from the 5' end of the probe is proportional to the target sequence concentration (copy number), and can be measured either at endpoint (post-amplification), or in real time', where the increase in emission intensity is followed on a per-cycle basis.
Alleles↗
Inhibition of HIV type 1 infectivity by constrained alpha-helical peptides: implications for the viral fusion mechanism.
Linear peptides derived from the membrane proximal region of the gp41 ectodomain are effective inhibitors of HIV type 1 (HIV-1)-mediated fusion events. These inhibitory peptides lack structure in solution, rendering mechanistic interpretation of their activity difficult. Using structurally constrained analogs of these molecules, we demonstrate that the peptides inhibit infectivity by adopting a helical conformation. Moreover, we show that a specific face of the helix must be exposed to block viral infectivity. Recent crystal structures show that the region of gp41 corresponding to the inhibitory peptides is helical and uses the analogous face to pack against a groove formed by an N-terminal coiled-coil trimer. Our results provide a direct link between the inhibition of HIV-1 infectivity by these peptides and the x-ray structures, and suggest that the conformation of gp41 observed by crystallography represents the fusogenic state. Other agents that block HIV-1 infectivity by binding to this groove may hold promise for the treatment of AIDS.
Amino Acid Sequence↗
Comparative analysis of the structure and function of the chicken c-myc and v-myc genes: v-myc is a more potent inducer of cell proliferation and apoptosis than c-myc.
To gain a more complete understanding of c-myc regulation in chickens, we have completed the structural characterization of the chicken c-myc gene and have begun to investigate c-myc transcription and protein expression. A comparison of c-myc structure and expression between mammals and birds presents an enigma: there are striking similarities in the pattern of gene expression in the absence of obvious sequence similarities in the controlling elements. We have begun to investigate c-myc and v-myc function using retroviral vectors that differ solely in the Myc proteins that they express. We show that while the overexpression of the smaller c-Myc protein is sufficient to induce morphological transformation in chicken embryo fibroblasts, overexpression of v-Myc provides a stronger signal for cells to enter the cell cycle and is a more potent inducer of apoptosis than c-Myc.
Amino Acid Sequence↗
Gene transfer and amplification in CHO cells. Efficient methods for maximizing specific productivity and assessment of genetic consequences.
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Animals↗
Plasmid integration, amplification and cytogenetics in CHO cells: questions and comments.
This paper addresses seven questions with respect to the transfer, integration, amplification and genetic stability of recombinant DNA in mammalian genomes. Most of these questions were raised with those issues in mind which are frequently discussed in the context of the manufacture of biologicals of therapeutic value on the basis of recombinant cell lines. For the most part, a high degree of ignorance has to be acknowledged and only very limited fragments of information are available. The reason for this ignorance is, as will become clear from this article, the sheer size and complexity of the mammalian genome and the inadequacy of presently available tools to unravel this complexity.
Animals↗
Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.
We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons.
Animals↗
Expression and role of c-myc in chondrocytes undergoing endochondral ossification.
To analyze the relationship between c-myc gene expression and chondrocyte proliferation and maturation during endochondral ossification, Day 18-19 chick embryo sterna were pulse-labeled with [3H]thymidine, and serial sections were processed for autoradiography and in situ hybridization. Proliferating chondrocytes, located in four distinct areas of the developing sternum, all contained high levels of c-myc transcripts, whereas postmitotic chondrocytes (such as hypertrophic chondrocytes) contained undetectable amounts. These findings were confirmed by Northern blot analysis and by the observation that antisense c-myc oligomer treatment inhibited proliferation in cultured chondrocytes. Constitutive overexpression of c-myc by retroviral vectors in immature chondrocyte cultures (c-myc cultures) maintained the cells in a proliferative state and blocked their maturation into hypertrophic chondrocytes. The lack of maturation in the c-myc cultures was corroborated by analysis of type X collagen gene regulation. Control immature cultures contained strong repressor activity for the type X collagen gene promoter, as revealed by transfection assays; repressor activity was lost upon maturation and activation of type X collagen synthesis. In the c-myc cultures, however, repressor activity persisted. Thus, c-myc participates in the normal changes in proliferation accompanying chondrocyte maturation in vivo and in culture. The decreases in c-myc expression and cell proliferation appear to be required for completion of maturation.
Animals↗
Appropriate in vivo expression of a muscle-specific promoter by using avian retroviral vectors for gene transfer [corrected].
The promoter regions of the chicken skeletal muscle alpha-actin (alpha sk-actin) and the cytoplasmic beta-actin genes were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Replication-competent retroviral vectors were used to introduce these two actin/CAT cassettes into the chicken genome. Chickens infected with retroviruses containing the alpha sk-actin promoter expressed high levels of CAT activity in striated muscle (skeletal muscle and heart); much lower levels of CAT activity were produced in the other nonmuscle tissues. In contrast, chickens infected with retroviruses containing the beta-actin promoter linked to the CAT gene expressed low levels of CAT activity in many different tissue types and with no discernible tissue specificity. Data are presented to demonstrate that the high levels of CAT activity that were detected in the skeletal muscle of chickens infected with the retrovirus containing the alpha sk-actin promoter/CAT cassette were not due to preferential infectivity, integration, or replication of the retrovirus vector in the striated muscles of these animals.
Actins↗
DNA methylation and oncogene expression in methapyrilene-induced rat liver tumors and in treated hepatocytes in culture.
Continued exposure of rats to carcinogenic doses of methapyrilene (MP) leads to elevated levels of 5-methyl-deoxycytidine (5MC) in liver DNA. Since gene expression often correlates with DNA methylation, we investigated these parameters in the MP-induced hepatocellular carcinomas of Fischer 344 rats. DNA was hypermethylated in liver tissue surrounding the tumors relative to liver tissue of untreated controls of the same age, while tumor DNA was not; DNA methylation declined to normal levels when MP treatment ceased. Gene expression analysis showed measurable levels of mRNA for c-Ki-ras, erb-B, erb-B2, hck, src, lyn, vav, trk, raf-1, l-myc, c-jun, c-yes, c-myc, c-abl, and p53. No significant differences in expression for these and other oncogenes were seen between tumors and surrounding livers, although erb-B2 and vav showed visible decreases compared with normal liver. Hypermethylation of DNA and expression of these oncogenes in MP-treated tissues were not correlated. Levels of mRNA for the same genes in MP-treated hepatocytes in culture were similar to in vivo levels; analysis of DNA synthesis levels showed that this gene expression pattern occurred in the absence of proliferation bursts or toxicity in these cells, thus suggesting that treatment in vivo may produce the same results.
Animals↗
Replication-competent retrovirus vectors for the transfer and expression of gene cassettes in avian cells.
We have constructed a series of replication-competent retrovirus vectors to introduce and express gene cassettes in avian cells. To characterize these vectors, we inserted the coding sequences for the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the chicken beta-actin gene promoter or the mouse metallothionein 1 gene promoter. In all cases, we found the structure of integrated proviruses to be stable during serial cell passage in vitro. Chloramphenicol acetyltransferase activity was detected biochemically and immunocytochemically in infected cells. Cassettes were inserted in the vectors in the same or in the opposite orientation with respect to viral transcription. Although both orientations were functional, the cassettes inserted in the forward orientation were usually expressed at higher levels than the corresponding backward constructions. The level of expression was strongly influenced by surrounding proviral sequences, particularly by the transcriptional enhancer elements within the retrovirus long terminal repeat sequences. Expression was higher with vectors that contained the polymerase (pol) region of the Bryan high-titer strain of Rous sarcoma virus. Inclusion of the Bryan pol region also improved vector replication in the chemically transformed quail fibroblast line QT6.
Animals↗
Vectors and genes for improvement of animal strains.
Strain improvement of agriculturally important animals will require efficient techniques for gene delivery, the ability to regulate the expression of the newly introduced genes and, most important, the identification of genes whose appropriate expression could cause improvement of the animal. We have developed a series of avian retroviral vectors that can be used to introduce new genetic information into the germ line of chickens, for which transgenics cannot be created by direct microinjection of DNA into fertilized eggs. We have identified a 220-bp segment of the chicken skeletal muscle alpha-actin gene that can cause other genes to be expressed specifically in striated muscle. This chicken promoter shows correct tissue specificity in transgenic mice and presumably could be used in other mammalian species. The skeletal muscle alpha-actin promoter has been inserted into the avian retroviral vectors and the promoter is functional in cultured cells infected by these retroviral vectors. The tissue specificity of the expression of the skeletal muscle alpha-actin promoter carried by the retroviral vectors will soon be tested in vivo. We are studying two types of genes that might be useful in strain improvement; genes that could produce dominant resistance to infection by pathogenic viruses, and genes that could play critical roles in muscle development. Expression of the envelope glycoprotein of retroviruses can specifically block the cellular receptor that viruses use to infect a susceptible cell. Expression of the avian leukosis virus subgroup A envelope in transgenic chickens prevents infection by pathogenic viruses of the same subgroup. We are attempting to block reticuloendotheliosis virus infection by expressing the reticuloendotheliosis envelope glycoprotein. We have shown that we can block infection in cultured cells, and we are now creating retroviral vectors for experiments in vivo. We have also begun to study the cellular homologue of the ski oncogene, which has been shown to stimulate the differentiation of quail myoblasts in vitro. Biologically active cDNAs have been isolated; we have now begun to analyse the effects of expressing the c-ski proteins in the whole animal.
Animals↗
The chicken skeletal muscle alpha-actin promoter is tissue specific in transgenic mice.
We have generated transgenic mouse lines that carry the promoter region of the chicken skeletal muscle alpha (alpha sk) actin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. In adult mice, the pattern of transgene expression resembled that of the endogenous alpha sk actin gene. In most of the transgenic lines, high levels of CAT activity were detected in striated muscle (skeletal and cardiac) but not in the other tissues tested. In striated muscle, transcription of the transgene was initiated at the normal transcriptional start site of the chicken alpha sk actin gene. The region from nucleotides -191 to +27 of the chicken alpha sk actin gene was sufficient to direct the expression of CAT in striated muscle of transgenic mice. These observations suggest that the mechanism of tissue-specific actin gene expression is well conserved in higher vertebrate species.
Actins↗
Helper-independent retrovirus vectors with Rous-associated virus type O long terminal repeats.
We have constructed nonpermuted replication-competent avian retrovirus vectors that derive from Rous sarcoma virus (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We describe here the construction and properties of corresponding vectors in which the long terminal repeats (LTRs) of the parental virus have been replaced by the LTRs of the endogenous chicken virus Rous-associated virus type O. The Rous-associated virus type O LTR vectors replicated approximately 1/10 as well as the parental vectors and expressed a test gene, chloramphenicol acetyltransferase, approximately 1/30 to 1/50 as well.
Animals↗
Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors.
We have previously described several helper independent vector constructions (S. Hughes and E. Kosik, Virology 136:89-99, 1984; J. Sorge and S. H. Hughes, J. Mol. Appl. Genet. 1:547-599, 1982; J. Sorge, B. Ricci, and S. Hughes, J. Virol. 48:667-675, 1983), all of which derive from Rous sarcoma virus. In this report we describe three improvements in the earlier constructions. First, the vectors have been restructured as proviruses, which considerably improves the efficiency of virus production following acute transfection. Second, a series of miniplasmids have been developed, which we call adaptors, and these miniplasmids can be used to convert virtually any DNA segment into a ClaI fragment suitable for insertion into the retroviral (or other) vectors. Adaptors have been developed that supply regions of functional significance, including a splice acceptor and an initiator ATG. Finally, the region of env defining subgroup specificity, A in the original vectors, has been substituted by the corresponding regions of subgroup B and D viruses, giving vectors with additional subgroup specificities and increased host ranges.
Animals↗
Methylation of the alphafetoprotein gene in cell populations isolated from rat livers during carcinogenesis.
We examined the methylation pattern and organization of the AFP gene in whole livers and in isolated cell populations purified from livers of rats fed a carcinogenic diet which interferes with DNA methylation. Using restriction endonuclease digestion, we find no differences in methylation pattern and overall organization of the AFP gene in oval cells (AFP-producers) and hepatocytes (non-producers) isolated at the early stages of carcinogenesis. Our studies indicate that in cell populations which produce AFP as well as in cells which are not active in AFP synthesis, the majority of the CCGG sites of the AFP gene are extensively methylated. In addition, we describe the existence of polymorphism in the AFP and albumin genes of Sprague-Dawley rats.
Albumins↗
Homology between rat liver RNA populations during development, regeneration, and neoplasia.
To investigate the degree of homology which may exist between rat liver RNA populations during development, regeneration, and neoplasia, we hybridized polyadenylated RNAs from (a) normal adult, (b) 24-h regenerating, (c) 20-day fetal livers, and (d) the transplantable Morris hepatoma 5123tc to homologous and heterologous complementary DNAs and to cDNAs enriched for sequences preferentially transcribed in either adult or fetal liver. We also compared the in vitro translation products of these RNAs. Analyses of normal adult, regenerating, and fetal liver RNA populations and their translation products show that the overall pattern of gene expression during liver regeneration differs little from that of normal adult rat liver and that mature hepatocytes do not appear to revert to an "immature" state upon reentering the cell cycle. Comparisons between fetal, normal adult, and tumor RNA populations revealed that RNA populations from fetal liver and the 5123tc tumor lack sequences normally expressed in the mature adult liver. However, the tumor does not "reexpress" sequences which are preferentially expressed in fetal livers.
Animals↗
Cell lineages in liver carcinogenesis: possible clues from studies of the distribution of alpha-fetoprotein RNA sequences in cell populations isolated from normal, regenerating, and preneoplastic rat livers.
We analyzed in isolated rat liver cell populations and in fetal and neoplastic livers the distribution of RNA sequences which hybridize with alpha-fetoprotein (AFP) complementary DNA clones. Parenchymal and nonparenchymal cell populations were isolated from normal, regenerating, preneoplastic, and bile duct-ligated rat livers. We found that oval cells, fetal liver, and a primary hepatocellular carcinoma contain the full length 2.3-kilobase AFP messenger RNA (mRNA); in normal adult rat liver, 2.3-kilobase AFP mRNA is found at low levels in an unidentified subpopulation of nonparenchymal cells but is not detected in hepatocytes; both parenchymal and nonparenchymal cells from normal or preneoplastic livers contain in variable proportion a smaller AFP RNA which hybridizes only with complementary DNA clones containing sequences located near the 5' end of the rat AFP gene; during liver regeneration induced by CCl4, elevation of the full length AFP mRNA occurs in nonparenchymal cells but seemingly not in hepatocytes. The results suggest that some cells in the nonparenchymal cell fraction of normal adult rat liver might retain the capacity to produce the 2.3-kilobase AFP mRNA found in large amounts in fetal livers, oval cells, and hepatic tumors. Although the nature of these cells remains to be determined, we suggest that such cells might be the source of the small amounts of AFP synthesized in normal rat liver and may constitute the proposed but as yet uncharacterized "facultative stem cell" compartment in rat liver.
Animals↗