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Biomedical subjects

G Khoury

Publications and source records attributed to G Khoury.

At least 91 records · Page 5Linked to original sources
PubMed1985

Stable transfer and restricted expression of a cloned class I gene encoding a secreted transplantation-like antigen.

The identification of a unique major histocompatibility complex class I gene, designated Q10, which encodes a secreted rather than a cell surface antigen has led to questions regarding its potential role in regulating immunological functions. Since the Q10 gene is specifically activated only in the liver, we sought to define the molecular mechanisms which control its expression in a tissue-specific fashion. Results obtained by transfection of the cloned Q10 gene, either in the absence or presence of a heterologous transcriptional enhancer, into a variety of cell types of different tissue derivations are consistent with the Q10 gene being regulated at two levels. The first is by a cis-dependent mechanism which appears to involve site-specific DNA methylation. The second is by a trans-acting mechanism which would include the possibility of an enhancer binding factor. The ability to efficiently express the Q10 gene in certain transfected cell lines offers an opportunity to obtain this secreted class I antigen in quantities sufficient for functional studies; this should also make it possible to define regulatory sequences which may be responsible for the tissue-specific expression of Q10.

Animals↗
PubMed1985

trans Activation of the simian virus 40 late transcription unit by T-antigen.

We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.

Animals↗
PubMed1984

JC virus enhancer-promoter active in human brain cells.

A human papovavirus, JCV, is the etiologic agent of the fatal demyelinating disease, progressive multifocal leukoencephalopathy. The JCV 98-base-pair tandem repeats, located to the late side of the viral replication origin, were shown to be a transcriptional regulatory element with enhancer-like activity in human fetal glial cells. These tandem repeats share significant homology with the 82-nucleotide rat brain-specific identifier RNA sequence.

Base Sequence↗
PubMed1984

Functional insertion of an Alu type 2 (B2 SINE) repetitive sequence in murine class I genes.

The regulation of expression of the family of MHC (major histocompatibility complex) class I genes is complex. Sequence analysis has revealed that class I genes from the H-2D subregion of the MHC (which includes the D and L genes) differ from the class I gene from the H-2K subregion (the K gene) by the insertion of a type 2 Alu-like repetitive element (the murine B2 sequence) within the 3' noncoding region of the D and L genes. The consequence of this insertion in the D and L genes is the introduction of a novel polyadenylation signal, which is preferentially used over the more distal signal, the analog of that found in the K gene. The insertion of the type 2 Alu-like sequence results in a change in the preferred site for endonucleolytic cleavage which is necessary for generating a correct 3' terminus for polyadenylation. The data demonstrate that the type 2 Alu-like sequence has a function; the data also suggest a possible regulatory role of this sequence in the expression of class I genes.

Animals↗
PubMed1984

Multiple enhancer domains in the 3' terminus of the Prague strain of Rous sarcoma virus.

The 3' terminal region of the Prague strain of Rous sarcoma virus (PrRSV) contains at least three distinct domains that comprise two functional enhancer elements. Two of these domains (designated B and C) are found in the U3 region of the 3' long terminal repeat (LTR) while the third (designated A) is located in the sequences immediately preceding the LTR termed XSR sequences. Combinations of adjacent domains [e.g., (A + B or B + C)] are capable of activating the expression of the SV40 early promoter (21 bp repeats and TATA box) coupled to coding sequences from the prokaryotic gene chloramphenicol acetyltransferase (CAT) while a single domain is inactive. Furthermore, duplication or triplication of the central domain B restores activity. The related, Schmidt-Ruppin, strain of RSV, contains an almost identical 3' LTR element, but differs in the enhancer sequences immediately preceding the 3' LTR. A model is presented in which the sequence differences may contribute to the difference in disease spectrum of transformation defective (td) variants of these viruses.

Animals↗
PubMed1984

Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen.

The early simian virus 40 (SV40) gene product, large tumor (T) antigen, is responsible for the initiation of viral DNA replication and the autoregulation of early gene expression through direct protein-DNA interactions. We investigated the role of T antigen in late viral gene expression, independent of its function in amplifying templates through DNA replication. SV40 DNA was transfected into BSC-1 and COS-1 cells and cultured in the presence of inhibitors of DNA replication. Electrophoretic immunoblot analysis indicated that both the onset and the extent of SV40 late gene expression is increased in COS-1 cells, which constitutively express SV40 T antigen. Blot hybridization analysis of poly(A)-selected RNA demonstrated that the level of synthesis of the major late structural protein VP-1 in COS-1 cells was due to increased transcription. Similar results were obtained when plasmids that contain the SV40 late gene but lack both the origin for viral DNA replication and the early gene coding region were transfected onto COS-1 cells. Using lines of SV40-transformed monkey kidney cells that express altered T antigens, we found that enhanced expression of the late gene product is correlated with the ability of T antigen to bind SV40 DNA. These results indicate that large T antigen plays a role in the stimulation of late viral gene expression.

Animals↗
PubMed1984

Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus.

A series of recombinant molecules were constructed which direct the expression of the easily assayed gene chloramphenicol acetyltransferase. We have used these recombinants to show that the 73/72-base-pair tandem repeat unit from the Moloney murine sarcoma virus long terminal repeat shares a number of properties with the prototypic enhancer element, the simian virus 40 72-base-pair repeat. Specifically, the Moloney murine sarcoma virus sequence significantly enhances the level of gene expression at both 5' and 3' locations and in either orientation relative to the test gene. It is able to enhance gene activity both from its own promoter and from a heterologous (simian virus 40) promoter. The 73/72-base-pair subunits of the Moloney murine sarcoma virus enhancer differ in sequence by four nucleotides and also in the strength of their enhancer function. The promoter distal A repeat is at least three times as active as the promoter proximal B repeat in enhancing chloramphenicol acetyltransferase expression. Results of these studies also show that the enhancer sequence alone is unable to induce gene activity but requires other promoter elements, including a proximal GC-rich sequence and the Goldberg-Hogness box.

DNA, Viral↗
PubMed1983

Comparison of class I (H-2) gene sequences. Derivation of unique probes for members of this multigene family.

The genes for the classical transplantation antigens are unique in that they belong to a multigene family of which each member is represented by a large number of alleles. Since all of these genes are highly related in sequence, it has been difficult to study the expression of individual members of this complex gene family. Based upon our initial suggestion that the 3' noncoding regions of these genes may be useful in identifying mRNA molecules transcribed from different loci, we have compared a large number of sequences from different inbred mouse strains and have been able to assign each of these sequences without ambiguity into distinct allelic series. Such accurate assignment has afforded the opportunity to compare the coding regions of these highly homologous genes and has led to the identification of sequences which are apparently unique to specific genes in the family. Synthetic oligonucleotides corresponding to each of the locus-specific unique regions have been used successfully to type a panel of cDNA sequences, as well as to quantitate the relative amounts of mRNA transcribed from distinct loci. The availability of these specific coding probes will allow the analysis of individual genes and their specific expression without interference from other highly homologous sequences in this multigene family.

Amino Acid Sequence↗
PubMed1983

BK viral enhancer element and a human cellular homolog.

Comparison of two closely related primate papovaviruses, simian virus 40 (SV40) and human BK virus (BKV), reveals that the only region of extensive divergence, the tandem sequences adjacent to the origins of DNA replication, is responsible in SV40 for enhancing early gene expression. This study demonstrates a similar enhancer function for the analogous repeated region in BKV. The dissimilarity in sequence of the BKV and SV40 enhancer elements suggests that they may have been acquired since SV40 and BKV diverged. A locus cloned from the human genome homologous to the BKV tandem repeats has been shown to function as low level enhancer element in mammalian cells. These data support the hypothesis that viral enhancer sequences may be evolutionarily related to host cell sequences.

Animals↗
PubMed1983

The human c-ras1H oncogene: a mutation in normal and neoplastic tissue from the same patient.

The c-ras1H oncogene can be distinguished from its normal cellular counterpart by the loss of a restriction endonuclease site. This sequence alteration is the basis of a rapid screening method for the presence of this oncogene. DNA's from 34 individuals were screened by this method, and all were homozygous for the normal allele. In contrast, DNA from a patient's bladder tumor, as well as DNA from his normal bladder and leukocytes, were heterozygous at that restriction endonuclease site. Further restriction enzyme mapping pinpointed the change in the mutant allele as being one of two nucleotides, either of which would change the 12th amino acid (glycine) in the normal c-ras1H gene product. Point mutations in the codon for this amino acid have previously been described in a bladder tumor cell line and in the viral oncogene v-rasH. These results indicate that the patient carried a c-ras1H oncogene in his germ line, raising the possibility that the c-ras1H oncogene confers a predisposition to neoplasia.

Base Sequence↗
PubMed1983

Secretion of a transplantation-related antigen.

Analysis of mouse cDNA clones has led to the identification of a class I (H-2)-related gene that encodes a truncated transplantation-like antigen. Unlike the products of the class I genes (H-2K, H-2D, and H2-L), which are synthesized and displayed on the surface of all cells, the class I-related gene product is expressed only in liver cells and is secreted. The region of the secreted molecule corresponding to the extracellular domain of the membrane-bound class I antigens shows unusual amino acid substitutions at positions otherwise invariably conserved. There is also loss of a glycosylation site that is used in all class I antigens. Within the region corresponding to the transmembrane domain are multiple nonconservative substitutions of hydrophobic residues, alterations that render the encoded protein incapable of inserting into the plasma membrane. Toward the end of the same domain, the polypeptide chain terminates abruptly and thus lacks the intracellular domain present on all class I antigens. A candidate for this secreted molecule, detected using various heteroantisera against class I antigens, has been identified. A potential role for this serum protein in mediating active tolerance is discussed.

Amino Acid Sequence↗
PubMed1983

Subcellular localization of the simian virus 40 agnoprotein.

The intracellular distribution of the simian virus 40 agnoprotein in infected cells was determined by indirect immunofluorescence and biochemical fractionation followed by indirect immunoprecipitation. The specific antibodies used in these studies were directed against either purified agnoprotein or a synthetic oligopeptide homologous to the N-terminus of the processed protein. Both procedures showed predominant localization of the agnoprotein to the cytosol and to the perinuclear region in association with the outer nuclear membrane. A minor but detectable fraction of the protein was also found in the nucleus. The definition of its subcellular distribution, as well as its high lability in vivo and affinity for nucleic acid, provide a basis for speculation on the function of this gene product.

Animals↗
PubMed1983

Mutant feline sarcoma proviruses containing the viral oncogene (v-fes) and either feline or murine control elements.

The sequences required for transformation by the Gardner-Arnstein (GA) strain of feline sarcoma virus (GA-FeSV) were defined by site-directed, in vitro mutagenesis of molecularly cloned proviral DNA. Portions of the Ga-FeSV provirus, subcloned in the plasmid pBR322, were mutagenized by deletion or frameshift at XhoI restriction sites flanking the nucleotide sequences presumed to encode the GA-FeSV transforming polyprotein (P108(gag-fes)). The biological activity of subgenomic and reconstructed full-genome-length molecules was assayed by transfection and focus induction in NIH 3T3 cells. Both mutant and wild-type molecules containing the intact P108(gag-fes) coding region induced foci of transformed cells at efficiencies between 10(4) and 10(5) focus-forming units per pmol of DNA; a deletion mutant lacking 3'-terminal v-fes sequences was completely nontransforming in parallel assays. Representative subcloned foci of transformed NIH 3T3 cells synthesized P108(gag-fes) with associated in vitro protein kinase activity. Focus-forming viruses could be rescued from transformed subclones induced by full-length proviral DNA, but not from cells transformed by subgenomic DNA lacking a 3' long terminal repeat (LTR). It was concluded that: (i) nucleotide sequences encoding P108(gag-fes) and its associated kinase activity are responsible for transformation, (ii) the GA-FeSV 3' env and LTR sequences are not required for focus induction, and (iii) the 3' LTR is necessary for rescue of infectious FeSV RNA. A chimeric DNA containing the 5' LTR and P108(gag-fes) coding region of GA-FeSV joined to the 3' LTR of Moloney murine sarcoma virus was both transforming and rescuable at high efficiency. Restriction analysis showed that passaged stocks of rescued transforming virus contained Moloney murine sarcoma virus U3 sequences at both proviral DNA termini, consistent with generally accepted models for LTR formation during reverse transcription. Wild-type GA-FeSV and the chimeric virus (here designated as GAHT), each rescued from NIH 3T3 cells with the same amphotropic murine leukemia virus, yielded approximately equal numbers of foci when titrated on CCL 64 mink cells. By contrast, on mouse NIH 3T3 cells, the focus-forming titer of GAHT was 1 to 2 log higher than that of FeSV. The foci induced on NIH 3T3 cells by GAHT appeared earlier and were reproducibly larger than those induced by GA-FeSV. Differences in transforming activity on NIH 3T3 cells were also found using colony formation in agar, showing that the more rapid appearance and larger size of foci formed in liquid media were not due to virus spread. These data suggest that transcriptional control signals within the viral LTR regulate the levels of the transforming gene product in a species-specific manner.

Animals↗
PubMed1983

Simian virus 40 tumor antigen: isolation of the origin-specific DNA-binding domain.

To localize the origin-specific DNA-binding domain on the simian virus 40 tumor (T) antigen molecule, we used limited proteolysis with trypsin to generate fractional peptides for analysis. A 17,000-Mr peptide was found to be capable of binding not only to calf thymus DNA, but also specifically to the simian virus 40 origin of DNA replication. This approximately 130-amino-acid peptide was derived from the extreme N-terminus of the T antigen and represented less than one-fifth of the entire molecule. The coding sequence for this tryptic peptide was located approximately between 0.51 and 0.67 map units (excluding the intron, which maps between 0.54 and 0.59). Since the first 82 amino acids are shared between large T and small t antigens, and since the latter does not bind DNA, it can be concluded that the sequence between isoleucine 83 and approximately arginine 130 is necessary for origin-specific binding by the T antigen. We also observed that in vivo phosphorylation of the T antigen within this region completely abolished the ability of the 17,000-Mr peptide to bind DNA. This observation is consistent with the idea that DNA binding by the T antigen is regulated by posttranslational modifications.

Animals↗
PubMed1982

Activation of SV40 genome by 72-base pair tandem repeats of Moloney sarcoma virus.

The simian virus 40 (SV40) 72-base pair (bp) tandem-repeated sequences have a crucial role as an activator element in viral gene expression. We replaced the SV40 72-bp repeat with a 72-bp repeat derived from the long terminal repeat (LTR) of cloned Moloney murine sarcoma virus (MSV) DNA. Although there is no detectable sequence homology to SV40, the MSV repeats can substitute functionally for the SV40 repeats and generate a viable virus in monkey kidney cells.

Animals↗