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Biomedical subjects

G Khoury

Publications and source records attributed to G Khoury.

At least 109 records · Page 6Linked to original sources
PubMed1982

Tissue-specific expression of an unusual H-2 (class I)-related gene.

Sequence analysis of mouse H-2 cDNA clones has suggested the existence of an unusual class of H-2 (class I)-related antigens that, unlike the classical membrane-associated molecules, retains only the extracellular portion and is likely to be secreted. The expression of this class of H-2-related mRNA is tissue restricted; it is detectable in liver but not in brain, kidney, testis, thymus, or spleen. In the liver, its accumulation represents about one-fourth of all the H-2 (class I)-specific transcripts. This class of transcripts is present in mice of different inbred strains, but the level of expression differs markedly among them. A model is presented in which such a soluble form of the H-2 antigen would play the role of a blocking factor in maintaining peripheral inhibition of H-2 recognition. This would ensure tolerance of the H-2 molecule as a self antigen while permitting it to act as a guidance molecule for the associative recognition of viral and tumor antigens by cytotoxic T cells.

Animals↗
PubMed1982

Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus.

The simian virus (SV40) 72-base pair (bp) tandem repeated sequences have recently been shown to function as activators or enhancers of early viral transcription. A recombinant viral genome was recently constructed by inserting 72-bp tandem repeats from the Moloney murine sarcoma virus (MSV) in place of the 72-bp repeats of SV40. Although this genome replicates in monkey kidney cells, its rate of large tumor antigen expression and replication is considerably slower than that of wild-type SV40. In mouse cells, however, equivalent levels of large tumor antigen appear to be expressed from both wild-type and recombinant genomes, suggesting a relationship between the level of enhancer activity and the host cell. To confirm this observation, we have applied a sensitive quantitative assay for gene expression based on the conversion of chloramphenicol to its acetylated forms. The gene encoding the enzymatic function chloramphenicol acetyltransferase was inserted into two vectors in which the enhancer sequences from SV40 or MSV were placed adjacent to the early SV40 promoter. The SV40 tandem repeats appear to activate gene expression to significantly higher levels in monkey kidney cells, but the MSV repeats are more active in two lines of mouse cells. These findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.

Acetyltransferases↗
PubMed1982

Mouse Lyt-2 antigen: evidence for two heterodimers with a common subunit.

Immunoprecipitation of [35S]methionine-labeled extracts of BALB/c thymocytes with mouse Lyt-2.2 monoclonal antibody yielded three components with subunit Mrs of 37,000, 32,000, and 28,000 on NaDodSO4/polyacrylamide gel in the presence of a reducing agent. Two-dimensional polyacrylamide gel analysis revealed that, in the absence of a reducing agent, the three polypeptide chains exist in the form of two heterodimers, each consisting of one molecule of a Mr 28,000 subunit covalently associated through disulfide bonds with either one Mr 37,000 subunit or one Mr 32,000 subunit. These two molecular structures are present in about equimolar ratios in the immunoprecipitate. Immunoautoradiographic analysis after electrophoretic transfer of proteins from a NaDodSO4/polyacrylamide gel to a nitrocellulose membrane indicated that the Lyt-2.2 determinant detected by the monoclonal antibody resides on the Mr 28,000 component, the common subunit of both heterodimeric structures. Lyt-2 precipitated from extracts of different T-cell growth factor-dependent cloned T-cell lines also showed similar structures, although the exact apparent Mrs of the respective components varied somewhat. The structure of the Lyt-2 antigen is of importance, particularly in the light of recent suggestions that it may be involved in the construction of one class of T-cell receptors.

Animals↗
PubMed1982

Discrete regions of simian virus 40 large T antigen are required for nonspecific and viral origin-specific DNA binding.

The nondefective adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses, Ad2+ND2 and Ad2+ND4, have been used to determine which regions of the SV40 genome coding for the large tumor (T) antigen are involved in specific and nonspecific DNA binding. Ad2+ND2 encodes 45,000 M4 (45K) and 56,000 Mr (56K) T antigen-related polypeptides. The 45K polypeptide did not bind to DNA, but the 56K polypeptide bound nonspecifically to calf thymus DNA, Ad2+ND4 encodes 50,000 Mr (60K), 66,000 Mr (66K), 70,000 Mr (70K), 74,000 Mr (74K), and 90,000 Mr (90K) T antigen-related polypeptides, all of which bound nonspecifically to calf thymus DNA. However, in more stringent assays, where tight binding to viral origin sequences was tested, only the 90K protein specified by Ad2A+ND4 showed specific high affinity for sequences at the viral origin of replication. From these results and previously published experiments describing the SV40 DNA integrated into these hybrid viruses, it was concluded that SV40 early gene sequences located between 0.39 and 0.44 SV40 map units contribute to nonspecific DNA binding, whereas sequences located between 0.50 and 0.63 SV40 map units are necessary for specific binding to the viral origin of replication.

Antigens, Viral↗
PubMed1981

Modification of SV40 T antigen by poly ADP-ribosylation.

The SV40 large T antigen is a multifunctional protein presumed to represent a single translation product of the early viral genes. A wide range of biological controls including the regulation of viral DNA replication, transcription and cell transformation has been attributed to T antigen. Previous evidence has indicated that large T antigen is modified in at least two ways, N-terminal acetylation and amino acid phosphorylation. In this study, we demonstrate a novel modification of a population of SV40 T antigen molecules by poly ADP-ribosylation. The covalent linkage of this oligonucleotide side chain to large T antigen, but not small t antigen, was demonstrated in experiments in which SV40-infected cells were labeled in vivo with 32P-orthophosphate or 14C-adenosine. Treatment of this labeled T antigen with snake venom phosphodiesterase released iso-ADP-ribose or treatment with ADP-ribose glycohydrolase released ADP-ribose. A method has been developed for the in vitro ADP-ribosylation of T antigen present in an infected cell nuclear extract with radiolabeled NAD. Since this type of modification is known to affect enzyme activity, its presence on T antigen suggests a similar role in the regulation of certain biological functions under the control of this protein.

Animals↗
PubMed1981

Expression of simian virus 40-rat preproinsulin recombinants in monkey kidney cells: use of preproinsulin RNA processing signals.

The complete rat preproinsulin gene I was cloned into a simian virus 30 (SV 40) vector. Most of the late region of the viral vector, including the SV40 intervening sequences (introns) and all of the major splice junctions, was deleted and replaced by the entire rat insulin gene. The recombinant molecules and a temperature-sensitive helper virus (tsA28) were inoculated into monkey kidney cultures. The formation of stable transcripts of the insulin insert was as efficient as the production of late SV40 mRNA. Analysis of these transcripts indicated that the rat preproinsulin gene nucleotide signals involved in RNA splicing and poly(A) addition were used. Examination of the 5' ends of the mRNAs showed several classes, one of which was the same size as the authentic rat insulinoma mRNA. This suggests that a portion of the transcripts may be initiated or processed faithfully, or both, at their 5' ends within rat insulin sequences. Significant quantities of a protein identified as rat proinsulin were synthesized. Detection of most of the proinsulin in the tissue culture medium suggests that this protein was secreted.

Animals↗
PubMed1981

Synthesis of stable unspliced mRNA from an intronless simian virus 40--rat preproinsulin gene recombinant.

We constructed a recombinant DNA molecule carrying the coding region and the 3' flanking sequences of the rat preproinsulin I gene, replacing the late region of a simian virus 40 (SV40) viral vector. The sequences making up the single intron of the preproinsulin I gene, located within the nontranslated leader region, as well as all of the 5' flanking sequences, have been deleted. The sequences remaining within the late transcriptional unit of SV40 do not retain a functional intron. This "intronless" mutant nevertheless directs the synthesis of substantial quantities of unspliced stable hybrid transcripts that are translated into polypeptides with insulin immunoreactivity. Two novel 5' termini, not used in wild-type SV40 transcripts, have been detected in the majority of the hybrid RNA molecules. These are located at analogous positions in both of the late SV40 72-base-pair repeats.

Animals↗
PubMed1981

Simian virus 40 tandem repeated sequences as an element of the early promoter.

On the late side of the simian virus 40 (SV40) DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base pairs long. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from which one of the 72-base-pair repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability, as recognized by the absence of early transcription and of tumor antigen production. These observations indicate that the 72-base-pair repeated sequences form an essential element in the early viral transcriptional promoter and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA. In a parallel experiment it was found that the extended deletion mutant was also unable to complement a late temperature-sensitive mutant of SV40, tsB. This suggests that the extended mutant is also defective in DNA replication or late transcription (or both).

Base Sequence↗
PubMed1981

p53 transformation-related protein: detection by monoclonal antibody in mouse and human cells.

A transformation-related protein of M(r) 53,000, designated p53, has been detected in a range of neoplastic cell types of the mouse by using immunoprecipitation of [(35)S]-methionine-labeled cell extracts with mouse antiserum [DeLeo, A. B., Jay, G., Appella, E., DuBois, G. C., Law, L. W. & Old, L. J. (1979) Proc. Natl. Acad. Sci. USA 76, 2420-2424]. We have now prepared a monoclonal antibody to p53 and have used it to study the occurrence and intracellular location of p53 by indirect immunofluorescence assays. In accordance with the results of immunoprecipitation, these tests showed p53 in all 13 transformed mouse cell lines studied. In each case, p53 was found in the nucleus. No p53 was detected in normal mouse fibroblasts, 3T3 cells, bone marrow cells, thymus cells, or embryo cells. A serologically related protein was detected in the nucleus of human cells by monoclonal antibody and was found in both normal and neoplastic cultured cells. Expression of p53 in human cells correlates with the growth characteristics of the culture, high p53 levels being associated with rapid cell proliferation and low p53 levels, with cessation of cell division. Normal and malignant human cells differ, however, with regard to the effect of confluency on p53 expression. Normal kidney epithelium and fetal brain cells, which express high p53 levels during exponential growth, show a prompt decrease in p53 associated with contact inhibition of cell division. Malignant cells, on the other hand, continue to express p53 after confluency and subsequent overgrowth of the monolayers. These results suggest that p53 may be involved in the normal regulation of cell division and that malignant transformation leads to abnormalities in the control of p53 expression.

Animals↗
PubMed1981

p53 transformation-related protein: detection of an associated phosphotransferase activity.

Malignant cells of the mouse transformed by a variety of different agents have been found to express high levels of a 53,000 Mr phosphoprotein (designated p53). Little or no p53 can be detected in normal mouse cells. The nucleus appears to be the predominant site of p53 localization in transformed cells. p53-related antigens are also found in transformed cells of rat, hamster, rabbit, and human. In cells transformed by simian virus 40 (SV40), p53 forms a complex with SV40 tumor (t) antigen, resulting in the coprecipitation of T antigen by monoclonal p53 antibodies. Immune complexes of p53 precipitated from extracts of SV40- or methylcholanthrene-transformed cells by monoclonal p53 antibodies have protein kinase activity. This enzymatic activity is dependent upon divalent cations, utilizing Mn2+ more effectively than Mg2+. The phosphorylation of p53 in this kinase reaction has been found to involve serine and threonine, but not tyrosine residues. In view of the finding that the transforming proteins of several different oncogenic viruses have kinase activity, the association of this activity with p53 is important with regard to the possibility of a common pathway of transformation by diverse agents.

Animals↗
PubMed1981

Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site.

With the premise that mRNAs transcribed in Escherichia coli from cloned eukaryotic DNA inserts do not possess the necessary regulatory signals for recognition by prokaryotic ribosomes, we have constructed a general plasmid vector carrying a chemically synthesized prokaryotic ribosome binding site that will ensure the efficient expression of eukaryotic proteins in E. coli. In addition to the regulatory signals necessary for ribosome recognition, the synthetic segment contains, at one end, a Pst I cleavage site which will direct its insertion to pBR322 DNA and, at the other end, a HindIII site to facilitate attachment of the passenger eukaryotic gene. Using simian virus 40 (SV40) tumor (t) antigen as a model system, we have ligated the SV40 DNA fragment containing the entire t antigen gene in tandem with the synthetic ribosome binding site to pBR322 DNA at the Pst I site, which lies within the coding sequence of the beta-lactamase gene. Initiation of transcription at the beta-lactamase promoter would produce a chimeric mRNA with the synthetic ribosome binding signals and the SV40 sequence flanked by beta-lactamase coding sequences. Utilization of the synthetic regulatory signals for initiation of translation is demonstrated by the efficient synthesis, in bacterial transformants, of authentic SV40 t antigen. Excision of the entire SV40 insert by HindIII from those clones that have retained intact HindIII sites at the junction between the ribosome binding site and the SV40 sequence would allow insertion of other heterologous DNAs by using HindIII linkers. The efficient expression of any DNA insert would require that the entire coding sequence be contiguous and that its termini be randomized by treatment with exonuclease III and nuclease S1 to vary the distance between the translational initiation codon and the synthetic ribosome binding site.

Antigens, Neoplasm↗
PubMed1981

Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector.

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.

Animals↗
PubMed1981

The regulation of SV40 gene expression in nonpermissive cells.

The limitations to SV40 growth in nonpermissive cells are poorly understood. In differentiated mouse cells, early mRNA and T-antigens are synthesized, but no viral DNA replication has been detected. A plausible explanation for the limitation to viral DNA synthesis in these cells might be the inability of a mouse cell-specific DNA polymerase to interact with the SV40 T-antigen-viral DNA complex. In spite of this abortive viral-cell interaction, SV40 late viral transcripts can be detected in infected mouse cells. Both early and late transcripts can be detected in infected mouse cells. Both early and late SV40 transcriptional activities peak between 10 and 15 h post infection; by 24 h after infection SV40 RNA is almost undetectable. In spite of the detection of late SV40 mRNA in mouse cells, we have been unable to detect any structural proteins (VP1, VP2, or VP3) encoded by these transcripts. A more restrictive interaction occurs between SV40 and undifferentiated mouse teratocarcinoma cell lines. Using an in vitro technique, the viral transcriptional complex (VTC) assay, we were able to demonstrate viral transcriptional activity on both the early and the late strands of SV40 in F9 embryonal carcinoma cells. Nevertheless, no mature processed mRNAs or viral encoded polypeptides were detected in these cells. After differentiation of the F9 cells with retinoic acid, however, spliced early mRNAs, as well as the SV40 T-antigen, were present.

Animals↗