Electrophoretic separation of both single- and double-stranded nucleic acids in the same urea-containing agarose gel.
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Blotting, Northern↗
Biomedical subjects
W Phares
Publications and source records attributed to W Phares.
Differential gene expression in cultured human Langerhans cells in response to phagocytic stimulation.
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Cells, Cultured↗
Spontaneous mutations in the human immunodeficiency virus type 1 gag gene that affect viral replication in the presence of cyclosporins.
Human immunodeficiency virus type 1 mutants that are resistant to inhibition by cyclosporins arise spontaneously in vitro during propagation in a HeLa-CD4+ cell line in the presence of a nonimmunosuppressive analog of cyclosporin A. Interestingly, the phenotype of all of the mutants examined is drug resistant and drug dependent, with both cyclosporin A and its analog. Four independently isolated mutants have been analyzed genetically by construction of recombinant proviruses in the NL4-3 parental strain background and subsequent testing of the chimeric viruses in HeLa cells. The cyclosporin-resistant, cyclosporin-dependent phenotype consistently transfers with a 1.3-kb fragment of gag, within which the four mutants share one of two possible single amino acid exchanges in a proline-rich stretch in the capsid domain of Pr55gag. These mutants provide the first evidence that mutations in human immunodeficiency virus type 1 gag confer resistance to cyclosporins; however, replication is conditional on the presence of the drug. In the T-cell line CEM, replication of the recombinant mutant viruses is also cyclosporin dependent. The drug-dependent replication in HeLa cells is stringent, and in the absence of cyclosporin only revertant viruses with the parental phenotype grow out of cultures infected with cyclosporin-dependent virus. In at least one isolate examined, the revertant phenotype appears to be due to suppressor mutations near the proline-rich region.
Amino Acid Sequence↗
Cyclosporine A-resistant human immunodeficiency virus type 1 mutants demonstrate that Gag encodes the functional target of cyclophilin A.
The cellular peptidyl-prolyl isomerase cyclophilin A is incorporated into human immunodeficiency virus type 1 virions via contacts with the proline-rich domain of the Gag polyprotein. Cyclosporine A and nonimmunosuppressive analogs bind with high affinity to cyclophilin A, compete with Gag for binding to cyclophilin A, and prevent incorporation of cyclophilin A into virions; in parallel with the disruption of cyclophilin A incorporation into virions, there is a linear reduction in the initiation of reverse transcription after infection of a T cell. Passage of human immunodeficiency virus type 1 in the presence of the drug selects one of two mutations, either of which alters the proline-rich domain of Gag and is sufficient to confer drug resistance on the cloned wild-type provirus. Neither mutation alters Gag's cyclophilin A-binding properties in vitro, and cyclophilin A incorporation into drug-resistant virions is effectively disrupted by cyclosporine A, indicating that the drug-resistant mutants do not require virion-associated cyclophilin A to initiate infection. That Gag's functional dependence on cyclophilin A can be differentiated genetically from its ability to bind cyclophilin A is further demonstrated by the rescue of a mutation precluding cyclophilin A packaging by a mutation conferring cyclosporine A resistance. These experiments demonstrate that, in addition to its ability to package cyclophilin A into virions, gag encodes the functional target of cyclophilin A.
Acquired Immunodeficiency Syndrome↗
The kappa B enhancer motifs in human immunodeficiency virus type 1 and simian virus 40 recognize different binding activities in human Jurkat and H9 T cells: evidence for NF-kappa B-independent activation of the kappa B motif.
The kappa B transcriptional enhancer motif, present in many viruses, is broadly active in many cell types. It is recognized by c-Rel/HIVEN86A in DNA affinity precipitation (DNAP) assays and by the Rel-related p50 and p65 subunits of the nuclear factor NF-kappa B in electrophoretic mobility shift assays (EMSA). We have analyzed activities that bind the human immunodeficiency virus type 1 and simian virus 40 kappa B motifs in two human leukemia cell lines, Jurkat and H9. In both DNAP and EMSA analyses of Jurkat cell extracts, we detected multiple kappa B motif-binding activities in addition to c-Rel/HIVEN86A and p50-p65 NF-kappa B. In Jurkat cell nuclear extracts, EMSA analysis revealed at least six specific DNA-protein complexes, of which one comigrated with the p50-p65 NF-kappa B complex. Formation of all six complexes was enhanced by stimulation of the cells with phorbol 12-myristate-13-acetate and phytohemagglutinin but was differentially affected by the salt concentration in the binding reaction and by the conditions of Jurkat cell growth. Nuclear extracts from both unstimulated and stimulated H9 cells revealed similar levels of five kappa B motif-specific complexes, all of which displayed mobilities distinct from those of the Jurkat cell complexes. Indeed, a complex corresponding to p50-p65 NF-kappa B was not detectable in nuclear extracts from unstimulated H9 cells although such a complex was apparent in nuclear extracts from stimulated H9 cells. In contrast to the inducibility of a p50-p65 NF-kappa B-like complex, transcriptional enhancers composed of multimerized kappa B motifs displayed similar high levels of activity in both the unstimulated and stimulated H9 cells. Thus, the activity of the kappa B motif in H9 cells corresponded to the abundance of the H9 cell-specific kappa B motif complexes and not to the levels of p50-p65 NF-kappa B complex. These results suggest that the broad activity of the kappa B enhancer element is not only due to the broadly distributed NF-kappa B activator but also to cell type-specific kappa B motif-binding activities.
Base Sequence↗
Functional similarities between human immunodeficiency virus type 1 and simian virus 40 kappa B proto-enhancers.
To search for broadly active enhancer elements within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we have used a proto-enhancer amplification assay. In this assay, the enhancer region of simian virus 40 (SV40) is replaced by heterologous regulatory sequences. Upon passage in African green monkey kidney cells. SV40 growth revertants can arise by amplification (usually duplication) of active protoenhancers within the heterologous sequences. Most of the HIV-1 U3 regulatory sequences were assayed; only amplification of one or both of the HIV-1 enhancer core kappa B motifs consistently resulted in viable SV40 virus. Examination of the cell-specific enhancer activity of the individual HIV-1 kappa B proto-enhancers showed that, like the broadly active SV40 kappa B proto-enhancer (C proto-enhancer), they are all active in noninduced cell lines of either lymphoid (H9 and Jurkat) or nonlymphoid (HeLa and CV-1) origin. Unexpectedly, one of three kappa B point mutants that exhibit little or no activity in unstimulated cells is as highly induced in stimulated Jurkat cells as are the wild-type kappa B proto-enhancers. This point mutation shows that kappa B-related proto-enhancers can display markedly different activation properties in unstimulated cells yet still activate transcription to similar levels in stimulated cells.
Animals↗
Transduction of proto-src sequences in tissue culture by a molecular clone of transformation-defective Rous sarcoma virus with an internal src deletion.
The sporadic appearance of nondefective (nd) Rous sarcoma virus (RSV) from cells in tissue culture infected with a molecular clone of transformation-defective RSV was examined. Southern analysis of extrachromosomal, virus-specific DNA of three independent ndRSV isolates in each case indicated restoration of an isogenic src by homologous recombination with cellular proto-src. The frequency of transduction was estimated by fluctuation analysis to vary between one transduction per 0.4 x 10(7) to 1.6 x 10(7) infected cells.
Animals↗
Viral oncogenes and cellular prototypes.
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Animals↗
Structural relationship between the chicken DNA locus, proto-fps, and the transforming gene of Fujinami sarcoma virus, delta gag-fps.
The avian Fujinami sarcoma virus (FSV) contains a hybrid transforming gene (delta gag-fps) with a 5' 1.3-kb portion derived from the gag gene of avian retroviruses and a 3' 2.8-kb portion (fps) derived from a cellular prototype. A lambda recombinant DNA clone carrying fps sequences within a 16-kb insert of cellular DNA, termed lambda proto-fps clone 12, has been selected from a chicken DNA library for comparison with the viral onc gene. Mapping of endonuclease-resistant proto-fps DNA fragments and hybridization with cloned viral DNA located FSV-related sequences at the 3' end of the insert within a region of about 4.25 kb. Alignment of endonuclease-resistant proto-fps and viral DNA fragments relative to common RNase T1-resistant oligonucleotide sequences of viral RNA, identified by fingerprinting DNA-RNA hybrids, indicated: (i) that proto-fps is colinear with viral fps but is interrupted by 1.75 kb of scattered sequences unrelated to viral fps; (ii) that among the nine endonuclease sites compared, proto-fps and viral fps share one PvuII, one BamHI, and possibly a Kpn1 site at homologous locations, and that they each have unique endonuclease sites and common sites at unique locations; (iii) that within 12 kb upstream from the 5' boundary of overlap with viral fps, proto-fps lacks gag-related sequences; and (iv) that proto-fps clone 12, like several others isolated by us, lacks at the 3' end an equivalent of the 3' 10 to 20% of viral fps. The eight endonuclease site-map coordinates of proto-fps and viral DNA also divided 44 fps oligonucleotides of viral RNA into 9 map segments. We conclude that the onc gene of FSV differs from proto-fps in delta gag and in multiple point mutations, compatible with a transforming function for the viral gene and a normal function for the cellular sequence homolog. Since proto-fps is unrelated to essential virion genes, the onc gene of FSV must have originated from cellular proto-fps by rare, illegitimate recombination.
Animals↗
The low tumorigenic potential of PRCII, among viruses of the Fujinami sarcoma virus subgroup, corresponds to an internal (fps) deletion of the transforming gene.
The avian sarcoma viruses FSV, PRCII, PRCIIp, and PRCIV share a related class of hybrid onc genes (delta gag-fps) defined by a specific nucleotide sequence fps and by delta gag-fps proteins of different sizes. Among these viruses, PRCII appears to have a lower tumorigenic potential than the others. Here we have compared fibroblast-transforming function and onc gene structure of these viruses. The fibroblast transforming ability of PRCII was lower than those of FSV, PRCIIp, and PRCIV. By gel electrophoresis the genomic RNA of PRCII measured 3.5 kb and those of FSV, PRCIIp, and PRCIV 4.5 kb; the delta gag-fps protein of PRCII measured 105 kilodaltons (kd), that of FSV 140 kd, and those of PRCIIp and PRCIV about 150 kd. By fingerprinting viral RNAs hybridized with molecularly cloned viral DNA the delta gag regions of PRCII and PRCIIp were defined to be 1.45 kb and that of FSV to be 1.3 kb. Fingerprint analysis of viral RNA-proto fps DNA hybrids showed the fps regions (approximately 2.8 kb) of FSV and PRCIIp to be isogenic. Compared to FSV and PRCIIp, the fps sequence of PRCII lacked a 1-kb region which maps between 0.3 and 1.3 kb from the 5' end of fps in FSV and PRCIIp. Based on oligonucleotide analysis, the shared fps complements of PRCII and PRCIIp were indistinguishable while that of FSV differed from those of the PRC viruses in scattered point mutations amounting to 1-2% of the RNA. Since all other regions of PRCII are isogenic with those of the highly tumorigenic variants PRCIIp, PRCIV, and FSV, it is concluded that the low fibroblast-transforming and oncogenic potential of PRCII reflects the internal fps deletion. Since the fps deletion reduces but does not eliminate transforming function, we suggest that the complete onc genes of viruses in the FSV subgroup include either several functional, or a regulatory and a functional fibroblast transforming domain. It has been reported that the 3' domains of the onc genes of viruses in the Fujinami subgroup and the onc genes of certain feline sarcoma viruses are distantly related. Since full transforming potential of the avian viruses depends on the 5' fps region not shared with the feline sarcoma viruses, we suggest that despite their structural homology, the avian and feline onc genes must have functionally different domains.
Animals↗
gamma-Glutamyltranspeptidase: a tumour cell marker with a pharmacological function.
The distribution of gamma-glutamyltranspeptidase activity (gamma GT) in adult, foetal, and neoplastic tissues in rats and humans is reviewed. The normal adult kidney, pancreas, and jejunum are high in this activity. Around the time of birth, gamma GT activity increases transiently in the liver, colon, and skin; many tumours of these organs often have elevated activity as well. Although this activity in tumour tissue may reflect a general expression of onco-foetal genes, subpopulations of cells having abnormally high gamma GT may have a selective growth advantage over their normal counterparts. We explore this latter possibility by considering the pharmacological function this enzyme may play. gamma GT may facilitate the detoxification of electrophiles by glutathione conjugation, so that cells can survive in an otherwise toxic environment. This growth advantage may be particularly important to the development of liver tumours in rats during chemical carcinogenesis.
Aging↗
Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells.
A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.
Alkaline Phosphatase↗
Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI beta-D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies.
Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and beta-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assay, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10(-5) M; the pH optimum for beta-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10(-5) M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for beta-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G1 through S and into G2-M phases of the cell cycle.
Animals↗
Anacystis nidulans mutants resistant to aromatic amino acid analogues.
Three classes of mutants of Anacystis nidulans were selected on the basis of resistance to fluorophenylalanine and 2-amino-3-phenylbutanoic acid. The most frequent type exhibited DAHP synthetase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate-D-erythrose-4-phosphate-lyase [pyruvate phosphorylating], EC 4.1.2.15) activity identical to that of the parental strain. The second type was characterized by extremely low levels of the activity. The third type had a DAHP synthetase showing decreased sensitivity to inhibition by L-tyrosine. The enzyme was purified 140-fold from wild-type and feedback-insensitive strains, and the kinetics of the reaction was examined. The activity of the wild-type enzyme was inhibited 75% in the presence of 2.0 X 10-3 M tyrosine, and the altered enzyme was inhibited 10%. The following apparent constants were obtained from kinetic studies with partially purified wild-type enzyme: S0.5 for D-erythrose-4-phophate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 1.4 X 10-4 M. Inhibition by tyrosine was mixed with respect to binding of both D-erythrose-4-phosphate and phosphoenolpyruvate. In addition, tyrosine promoted cooperative interactions in the binding of phosphoenolpyruvate. For the altered enzyme the following apparent constants were obtained: S0.5 for D-erythrose-4-phosphate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 2.9 X 10-4 M. Inhibition by tyrosine was mixed with respect to D-erythrose-4-phosphate and competitive with respect to phosphoenolpyruvate. Tyrosine did not promote cooperative effects in the binding of phosphoenolpyruvate to the altered enzyme.
4-Aminobutyrate Transaminase↗