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S R Tronick

Publications and source records attributed to S R Tronick.

At least 37 records · Page 2Linked to original sources

rev-like transcripts of caprine arthritis encephalitis virus.

The pattern of expression of the caprine arthritis encephalitis virus genome (CAEV) in acutely infected tahr lung cells was found to be complex and temporally regulated. Employing Northern analysis, five CAEV-specific transcripts, 9, 6.5, 5.0, 2.5, and 1.4 kb, were detected. Nucleotide sequence analysis established the genetic structure of two species of cDNA, isolated from a library of CAEV-infected tahr cells, and suggested that they represent rev-like transcripts. One of these cDNA species was composed of three exons--the leader, an exon derived from the 5' region of env, and an exon which spanned the 3' orf. The second cDNA species consisted of four exons--three of which were identical with those of the former species. The additional exon (the second) was located at the 3' end of pol. These transcripts could potentially encode three proteins--a Rev-like protein, which is a fusion of 38 amino acids derived from the N-terminus of env and 91 residues from the 3' orf; a truncated form of the env transmembrane protein, and a novel protein, designated X composed of 73 amino acids. Thus, CAEV, like other lentiviruses, displays a complex pattern of gene expression, characterized by alternative splicing and the production of potentially polycistronic transcripts.

Amino Acid Sequence↗

Alternative splicing generates at least five different isoforms of the human basic-FGF receptor.

Fibroblast growth factors (FGFs) are polypeptide mitogens that induce the proliferation of a wide variety of cell types. Of the seven family members, the best characterized are basic and acidic FGF. In addition to their mitogenic effects, they participate in angiogenesis, differentiation and maintenance of survival of neurons, cell migration and embryonal development. Of all family members, keratinocyte growth factor (KGF) is unique in that it is a specific mitogen for epithelial cells and does not interact with the FGF receptor of fibroblasts. To study the interactions between KGF and its receptor, we isolated KGF and FGF receptors from keratinocytes and fibroblasts, respectively. In the course of this study, we isolated five different variants of the FGF receptor from human fibroblasts and showed that all were derived from a single genetic locus. Four of these variants encode transmembrane receptors and can be divided into two subgroups that differ from one another with respect to the number (two or three) of immunoglobulin (Ig)-like domains. Within each subgroup, one receptor differed from the other by the presence of a two-codon insertion. Thus, all the variations among the four isoforms are localized to their ligand binding domains. The fifth isoform encodes a molecule truncated just 3' to the first Ig-like domain and thus could be secreted from the cell. The transcripts encoding the long and short isoforms were found to be expressed in many cell types, but their relative levels of expression varied greatly depending on the cell type. These findings indicate that alternative splicing generates diverse FGF receptor isoforms in human cells.

Amino Acid Sequence↗

A region of proto-dbl essential for its transforming activity shows sequence similarity to a yeast cell cycle gene, CDC24, and the human breakpoint cluster gene, bcr.

Proto-dbl is a human proto-oncogene, whose oncogenic activation was initially detected by DNA transfection. We report significant sequence similarity between the predicted proto-dbl product and the products of CDC24, a Saccharomyces cerevisiae cell division cycle gene required for correct budding and establishment of cell polarity, and bcr, a gene implicated in the pathogenesis of chronic myelogenous leukemia (CML). Of 925 residues of the predicted proto-dbl protein, a stretch of 238 residues showed 29% and 22% identity over a region of similar length of the CDC24 and bcr proteins, respectively. When evolutionarily conservative substitutions were taken into account, the similarities were 68.8% and 71.6% for proto-dbl/CDC24 and proto-dbl/bcr gene products, respectively. Moreover, all three sequences were predicted to be markedly hydrophilic over this region. Very small deletions within the conserved region completely abolished transforming activity of dbl, while extensive deletion outside of this region had no effect. Even substitutions over a small stretch of close similarity with the other proteins substantially impaired transforming activity. Cells transformed by the dbl oncogene, like cdc24 mutants arrested at the nonpermissive temperature, form multinucleate cells. Thus, our findings indicate that the conserved region is an essential domain that may reflect important functional similarities among these otherwise highly divergent molecules.

Amino Acid Sequence↗

Identification of sequences encoding the equine infectious anemia virus tat gene.

Equine infectious anemia virus (EIAV), a lentivirus, encodes a trans-activator (tat) which stimulates gene expression directed by the viral long terminal repeat (LTR). This function has been previously shown by us and others to be encoded by sequences within the middle region of the EIAV genome in which two short open reading frames, S1 and S2, reside. In the present study, by using in vitro mutagenesis, we show that disruption of S1, but not S2, completely abolished trans-activation. Addition of oligonucleotides complementary to S1 to cells transfected with a tat expression vector resulted in inhibition of trans-activation. EIAV cDNAs were isolated from a library of EIAV-infected cells constructed by using a eukaryotic expression vector. One cDNA clone which contained S1 sequences was able to trans-activate the EIAV LTR. Sequence analysis of this cDNA clone revealed that, in addition to S1, two other open reading frames were present. The cDNA still retained its activity when the latter two sequences were deleted.

Amino Acid Sequence↗

An in-vivo infectivity assay for cloned retroviruses lacking a susceptible cell culture.

The lymphoproliferative disease virus (LPDV) of turkeys is the retroviral agent of etiology of a rapidly developing, naturally occurring, lymphoproliferative process. Recently we have molecularly cloned the viral genome. The lack of a susceptible cell culture which can sustain LPDV replication hampered the analysis of the infectious capability of the cloned genome. Based on the efficient in-vivo replication of LPDV we have developed a sensitive in-vivo approach aimed at establishing the infectious capability of the cloned provirus. According to this approach, peripheral leukocytes withdrawn from 3-week-old turkeys were transfected with the cloned DNA and the transfected leukocytes were re-injected into the turkey from which they had been obtained. The injected leukocytes enabled the efficient expression of the viral genome and the release into the blood stream of LPDV virions, which thereafter could travel to their appropriate in-vivo target lymphoid cells and start multiple replication cycles, resulting in the development of a detectable viremia. The applicability of this in-vivo assay for other cloned viral genomes is discussed.

Animals↗

Pattern of transcription of the genome of equine infectious anemia virus.

The pattern of expression of the equine infectious anemia virus (EIAV) genome in a persistently infected canine cell line was determined. Five EIAV-specific transcripts (8.2, 5.0, 4.0, 2, and 1.8 kilobases [kb]) were detected by using subgenomic restriction enzyme fragments of EIAV DNA and EIAV-specific oligonucleotides as probes. The 8.2-kb mRNA could be shown to represent viral genomic RNA, whereas the smaller transcripts were generated by splicing events. Evidence was obtained that indicated that each subgenomic RNA species shared a common 5'-splice donor. The 5.0-kb mRNA was found to be expressed at relatively low levels, was difficult to detect consistently, and appeared to be generated by a single splicing event which linked the 5' exon to the 3' region of pol. The 4.0-kb transcript was concluded to be the env mRNA on the basis of its hybridization pattern with the various probes and its abundance. The 2-kb species was found to be multiply spliced and was encoded by sequences derived from orf2 but was not detected by probes representing 3'-env/3'-orf sequences. The 1.8-kb species was shown to consist of sequences representing orf1, part of orf2, and the 3'-orf/env and may represent the message for the EIAV trans-activator gene.

Amino Acid Sequence↗

Chromosomal localization of DBL oncogene sequences.

The DBL oncogene was generated by rearrangements involving three discontinuous regions of the human genome. Analyses of panels of human X rodent somatic cell hybrids demonstrated that the DBL proto-oncogene located on the X chromosome (just proximal or distal to bands q26-27.2) underwent recombination at its 5' and 3' ends with sequences derived from chromosomes 3 (p13q-ter) and 16 (p13-q22), respectively. DBL was localized to chromosome Xq27-q28 by in situ hybridization. Another oncogene, MCF2, was previously shown to contain sequences derived from Xq27 as well. Comparison of the restriction maps and nucleotide sequences of genomic and cDNA clones representing the chromosome X-specific sequences of the DBL oncogene and MCF2, taken together with their chromosomal localization, indicates that these oncogenes were derived from the same genetic locus.

Animals↗

Development of an enzyme-linked immunosorbent assay for equine infectious anemia virus detection using recombinant Pr55gag.

To provide more sensitive and convenient methods for the detection of equine infectious anemia virus (EIAV), we developed an enzyme-linked immunosorbent assay (ELISA) employing the EIAV gag precursor (Pr55gag) produced by using recombinant DNA techniques. The antigenic reactivity of the recombinant EIAV Pr55gag was found to be equivalent to that of the virion p24gag and elicited high-titered antiserum in rabbits. When a large number of horse sera were analyzed for the presence of antibodies to EIAV by this ELISA, a radioimmunoassay for EIAV p15gag, or the standard agar gel immunodiffusion test, there was 98.7% concordance among the assays. By using the ELISA it was possible to specifically detect antibodies earlier after experimental infection of horses with EIAV than with the other two tests. A competition ELISA developed in order to detect EIAV gag antigens was found to be approximately 15 times more sensitive than the radioimmunoassay for EIAV p15gag. Antigens of other animal lentiviruses as well as those of the prototype oncovirus failed to compete in this assay.

Animals↗

Analysis of regulatory elements of the equine infectious anemia virus and caprine arthritis-encephalitis virus long terminal repeats.

We analyzed the equine infectious anemia virus (EIAV) long terminal repeat (LTR) for sequences that influence its promoter activity and ability to be trans-activated by the EIAV tat gene product. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 (SV40) regulatory sequences were used for these studies. We were able to identify the EIAV promoter region and showed that sequences within the U3 region significantly inhibited LTR-directed transcription. However, when placed in a heterologous context (SV40 promoter) these U3 sequences functioned as an enhancer. trans-activation of the EIAV LTR was found to depend upon sequences downstream of the transcription initiation site and also within U3. Deletion mutagenesis experiments showed that the major downstream element was present in a 46-nucleotide stretch (+4 to +50). An SV40 promoter construct containing these sequences could be trans-activated in cells expressing the EIAV tat gene product. For comparative purposes we also examined the LTR of another animal lentivirus, caprine arthritis-encephalitis virus (CAEV), for positive and negative transcriptional regulatory elements and demonstrated the presence of an enhancer within its U3 sequence. There is evidence that trans-activation of the CAEV LTR requires U3 sequences. When the EIAV U3 region was replaced by the CAEV U3 sequence, the promoter activity of the EIAV LTR was markedly elevated, but the responsiveness to the EIAV trans-activator could not be restored.

Animals↗

Molecular cloning of an oncogenic replication-competent virus that causes lymphoproliferative disease in turkeys.

The lymphoproliferative disease virus of turkeys was molecularly cloned, structurally mapped, and shown to represent a distinct class of retroviruses evolutionarily related to the avian leukemia-sarcoma virus group. The cloned provirus did not contain any known oncogene or other cellularly derived sequences and was established as a replication-competent oncogenic entity capable of inducing the disease in the absence of any associated transforming counterpart.

Animals↗

Molecular cloning and characterization of the human dbl proto-oncogene: evidence that its overexpression is sufficient to transform NIH/3T3 cells.

We isolated cDNA clones representing the human dbl proto-oncogene transcript. Nucleotide sequence analysis revealed an open reading frame encoding a predicted protein of 925 amino acids. Using peptide antisera directed against specific proto-dbl peptides, a 115-kd protein was detected in COS cells transfected with an expression vector containing the entire coding region of proto-dbl. This mol. wt is consistent with that predicted from the open reading frame. We have previously shown that the dbl oncogene was generated by substitution of the 5' portion of proto-dbl with an unrelated human sequence. In this study we show that this rearrangement resulted in the loss of the 497 amino-terminal codons of the dbl proto-oncogene. Under the influence of a strong promoter proto-dbl could readily transform NIH/3T3 cells but its transforming activity was less than that of the dbl oncogene driven by the same promoter. Proto-dbl overexpression is, therefore, sufficient to transform NIH/3T3 cells, but specific structural alterations of its coding region significantly enhance its transforming activity. No apparent similarity was detected between the predicted proto-dbl product and other known proto-oncogenes. However, a stretch of 300 amino acids within the N-terminal half of proto-dbl showed structural similarity to the intermediate filament vimentin. This region in proto-dbl contains a heptad repeat motif characteristic of an alpha-helical coiled-coil structure. Taken together, these findings indicate that the human proto-dbl represents a new class of cellular oncogenes that may be related to cytoskeletal elements of the cell.

Amino Acid Sequence↗

The predicted DBL oncogene product defines a distinct class of transforming proteins.

The DBL transforming gene was originally identified by transfection of NIH 3T3 cells with DNA from a human B-cell lymphoma. This gene was found to have arisen as a result of recombination of the 3' portion of the DBL protooncogene coding sequences with an unrelated segment of human DNA. It encodes a cytoplasmic protein that is equally distributed between cytosol and crude membrane fractions. To further characterize this transforming gene, a biologically active cDNA clone of the DBL transforming gene mRNA was isolated. Analysis of the sequence of the DBL oncogene cDNA revealed a long open reading frame that encodes a hybrid protein whose first 50 amino acids (at least) derive from a complete exon of a different locus. No significant homology with known oncogenes or any known protein sequences was demonstrated. The computer analysis of the predicted DBL protein indicated it is highly hydrophilic with no hydrophobic domains characteristic of a membrane-spanning region or signal peptide. Thus, the DBL oncoprotein is distinct among known transforming gene products.

Amino Acid Sequence↗

Localization of sequences responsible for trans-activation of the equine infectious anemia virus long terminal repeat.

We used the Escherichia coli chloramphenicol acetyltransferase gene (cat) to study sequences that influence expression of the equine infectious anemia virus (EIAV) genome. The EIAV long terminal repeat (LTR) directed CAT activity in a canine cell line, but at levels much lower than those achieved with other eucaryotic viral promoters. In the same cells infected with EIAV or cotransfected with molecularly cloned EIAV genomic DNA, LTR-directed activity was markedly enhanced. Comparison of cat mRNA and protein levels in these cells indicated that this trans-activating effect could be accounted for by a bimodal mechanism in which both transcriptional and posttranscriptional events are enhanced. trans-Activation but not promoter activity was abolished by deletion of the R-U5 region of the EIAV LTR. EIAV sequences responsible for the trans-activating function could be localized to a region encompassing the 3' and 5' termini of the pol and env genes, respectively (nucleotides 4474 to 5775). Interestingly, this stretch harbors a short open reading frame with some amino acid sequence similarity to the human immunodeficiency virus type I tat gene product.

DNA Mutational Analysis↗

Primary structure of the human fgr proto-oncogene product p55c-fgr.

Normal human c-fgr cDNA clones were constructed by using normal peripheral blood mononuclear cell mRNA as a template. Nucleotide sequence analysis of two such clones revealed a 1,587-base-pair-long open reading frame which predicted the primary amino acid sequence of the c-fgr translational product. Homology of this protein with the v-fgr translational product stretched from codons 128 to 516, where 32 differences among 388 codons were observed. Sequence similarity with human c-src, c-yes, and fyn translational products began at amino acid position 76 of the predicted c-fgr protein and extended nearly to its C-terminus. In contrast, the stretch of 75 amino acids at the N-terminus demonstrated a greatly reduced degree of relatedness to these same proteins. To verify the deduced amino acid sequence, antibodies were prepared against peptides representing amino- and carboxy-terminal regions of the predicted c-fgr translational product. Both antibodies specifically recognized a 55-kilodalton protein expressed in COS-1 cells transfected with a c-fgr cDNA expression plasmid. Moreover, the same protein was immunoprecipitated from an Epstein-Barr virus-infected Burkitt's lymphoma cell line which expressed c-fgr mRNA but not in its uninfected fgr mRNA-negative counterpart. These findings identified the 55-kilodalton protein as the product of the human fgr protooncogene.

Amino Acid Sequence↗

Nucleotide sequence analysis of equine infectious anemia virus proviral DNA.

The nucleotide sequence of the integrated form of the genome of the equine infectious anemia virus was determined. By comparison with LTR sequences of other retroviruses, signals for the control of viral gene transcription and translation could be identified in the EIAV LTR. Open reading frames for gag and pol genes were identified and their sequences matched very closely to those determined previously by others. However, in the present study, the pol gene reading frame was open throughout its entire length. The open reading frame for the env gene product was constructed from the sequences of two independent EIAV clones. Thus, a noninfectious genomic-length clone was shown to contain a frameshift mutation approximately in the middle of the presumed env gene coding sequence, whereas the sequence of another clone was open in this region. The deduced amino acid sequences of the EIAV gag and pol products showed closer evolutionary relationships to those of known lentiviruses than to other retroviruses. There was also partial sequence homology between predicted env gene products of EIAV, visna virus, and HTLV-III/LAV. Sequences analogous to the sor region of other lentiviruses could not be identified in our EIAV clone. A short open reading frame at the 3' end of the genome that overlapped env but not the 3' LTR was present but lacked significant sequence similarity to the 3' open reading frames of other lentiviruses. Thus, the sequence and general structure of EIAV most closely resemble those of known lentiviruses.

Amino Acid Sequence↗

Independently activated dbl oncogenes exhibit similar yet distinct structural alterations.

The dbl oncogene was initially isolated following transfection of NIH3T3 cells with DNA of a human diffuse B cell lymphoma. Its transcribed sequences were shown to be distributed over a 30-kb span within a molecularly cloned 45-kb segment of human DNA which contained the transforming gene. By restriction mapping, its transcribed region corresponded to that of its normal allele, except at the 5' end where a rearrangement involved transcribed dbl oncogene sequences from another locus. An independent isolate of a dbl-related transforming gene was obtained following transfection of NIH3T3 cells with DNA of a human nodular poorly differentiated lymphoma (NPDL). Physical mapping indicated that this transforming gene, designated NPDL-dbl, shared considerable homology with the dbl oncogene, but differed at both 5' and 3' termini. Its point of divergence from the normal allele at the 5' end was at least 10 kb upstream from that of the dbl oncogene. The oncogenes each expressed truncated transcripts compared to the 5.3-kb normal transcript. The dbl and NPDL-dbl oncogene translational products of 66 and 76 kDa, respectively, were consistent with their corresponding major 2.8- and 3.5-kb transcripts. It was not possible to detect evidence of the 5' structural rearrangements associated with these oncogenes in either of the original tumors. Thus, if these rearrangements were critical to their activation, they occurred in the process of gene transfer or in vivo in only a minority of tumor cells.

Animals↗