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S R Tronick

Publications and source records attributed to S R Tronick.

At least 19 recordsLinked to original sources
PubMed1996

Evidence for the involvement of the Wnt 2 gene in human colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent cancers in humans. It develops via a multistage process involving alterations of both protooncogenes and tumor suppressor genes. In the present report we determined the level of expression of several Wnt genes in CRC by RT-PCR and direct sequencing. While Wnt-1 was not detectably expressed in any colonic tissues, Wnt-5a gene was efficiently expressed both in nontumorous as well as in colonic tumor tissues. In contrast, the Wnt-2 gene, which was expressed at low levels in normal colon, exhibited overexpression in all tumor tissue samples at the different Dukes' stages of CRC progression, including premalignant polyps and liver metastases. Overexpression of the Wnt-2 gene occurred also in other digestive neoplasms such as gastric and esophageal carcinomas, as well as in diverticulitis associated with stenosis or pseudo-tumor.

Base Sequence↗
PubMed1996

BRCA1 is secreted and exhibits properties of a granin.

Germline mutations in BRCA1 are responsible for most cases of inherited breast and ovarian cancer. However, the function of the BRCA1 protein has remained elusive. We now show that BRCA1 encodes a 190-kD protein with sequence homology and biochemical analogy to the granin protein family. Interestingly, BRCA2 also includes a motif similar to the granin consensus at the C terminus of the protein. Both BRCA1 and the granins localize to secretory vesicles, are secreted by a regulated pathway, are post-translationally glycosylated and are responsive to hormones. As a regulated secretory protein, BRCA1 appears to function by a mechanism not previously described for tumour suppressor gene products.

Amino Acid Sequence↗
PubMed1996

Two species of Rev proteins, with distinct N termini, are expressed by caprine arthritis encephalitis virus.

Several cDNA clones representing alternatively spliced Rev-specific transcripts were isolated from a cDNA library prepared from Himalayan tahr cells infected with caprine arthritis encephalitis virus (CAEV). We previously characterized two rev-like cDNA species, d1 and d2, and a tat e1 cDNA containing the rev coding sequence downstream to the tat. In these cDNAs, the rev coding domain derives its amino terminus from the N terminus of env, which is spliced to the 3' open reading frame encoding the putative Rev protein. In this study, we report the genetic structure of a fourth rev-like cDNA (designated g1), which lacks the 5' env-derived sequences. All of these rev transcripts, including cDNA g1, increased the level of chloramphenicol acetyltransferase expression when cotransfected with a reporter plasmid containing the CAEV Rev-response element-spanning region downstream of the cat coding sequences. Western blot (immunoblot) analysis showed that each transfected cDNA species gave rise to a 16-kDa protein lacking env-encoded amino-terminal epitopes. In contrast, CAEV-infected Himalayan tahr cells expressed only a 20-kDa protein, whose N terminus, in contrast, is derived from the env. Moreover, only the 20-kDa protein was also detected in the mature CAEV virions. These observations suggest that the transcripts d1, d2, and e1 can potentially, in appropriate cellular context, encode two Rev isoforms differing in their N termini, whereas the g1 transcript encodes only the 16-kDa species. Elucidation of the significance of the 16-kDa Rev protein in CAEV biology must await further studies.

Amino Acid Sequence↗
PubMed1995

Different fibroblast growth factor 1 (FGF-1) transcripts in neural tissues, glioblastomas and kidney carcinoma cell lines.

We have previously reported the tissue specific distribution of four different FGF-1 transcripts containing alternative 5' untranslated exons spliced to the first protein coding exon. The predominant transcript in brain is FGF-1.B and in kidney FGF-1.A. Others have shown, by in situ hybridization and immunohistochemical analysis, that expression of FGF-1 in the brain is exclusively in neural cells but not in glial cells. Here we have examined the distribution of FGF-1.B and FGF-1.A transcripts in glioblastoma and retinal tissues and in kidney carcinoma cell lines. Our results show that FGF-1.B is the predominant transcript in neural derived tissues including both the diabetic retina and normal retina tissues. Surprisingly, FGF-1.B transcript is highly expressed in glioblastoma tissues. In contrast, a normal brain glial cell line, CHII, expresses very low levels of FGF-1 mRNA. These results strongly implicate the role of FGF-1 in the etiology of glioblastoma. We also examined several kidney carcinoma derived cell lines for the expression of FGF-1 mRNA. Most of these kidney cell lines do not express any FGF-1 transcripts. An interpretation by deduction is that kidney adenocarcinomas are derived from cortex but medulla has been reported as the site of FGF-1 synthesis. Of the kidney derived cell lines which are positive for FGF-1 message, only one expressed FGF-1.A transcript. The data may suggest that the establishment of kidney cell lines results in a switch of promoter usage from the 1.A seen in kidney tissue. Similarly, culturing of glioma cell lines may result in a switch from FGF-1.B seen in glioma tissues to FGF-1.D seen in most glioma cell lines. Continued studies of the FGF-1 transcripts, their functional promoters and their tissues distribution will provide insight into the potential role of FGF-1 in cell growth, tissue differentiation and malignant transformation.

Alternative Splicing↗
PubMed1995

Identification of sequences in the long terminal repeat of the lymphoproliferative disease virus required for efficient transcription.

We analyzed the long terminal repeat (LTR) of the lymphoproliferative disease virus of turkeys for sequences that influence its promoter activity by using the chloramphenicol acetyltransferase assay. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 regulatory sequences were used for these studies. Through transfection experiments, we identified a negative regulatory element residing at the 5' end of the U3. The two imperfect direct repeats (DRs) located at nt - 170 to - 125 upstream of the RNA transcription site were identified as enhancer elements which could stimulate transcription of a heterologous promoter in an orientation independent manner. Specific interaction of nuclear factors with the DRs element was identified. The two DRs contain cArg motifs which are suggested to play a role in tissue specific expression of several cellular genes.

Alpharetrovirus↗
PubMed1994

Biological activity and intracellular location of the Tat protein of equine infectious anemia virus.

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10(11) bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by 'scrape loading'. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.

Amino Acid Sequence↗
PubMed1994

Genome organization of a biologically active molecular clone of the lymphoproliferative disease virus of turkeys.

The lymphoproliferative disease retrovirus (LPDV) induces an acute, horizontally transmitted disease of turkeys that is often fatal. Although LPDV cannot be grown in cultured cells, it was possible to isolate molecular clones of biologically active integrated proviral genomes from spleens of infected turkeys. Based upon molecular hybridization and nucleotide sequence comparisons of its pol gene, LPDV was shown to represent a distinct group of avian retroviruses most closely related to avian sarcoma-leukemia viruses. Here we report the complete nucleotide sequence of the LPDV genome as well as amino acid sequence analysis of its gag gene products. The genetic organization of LPDV is characteristic of members of the oncovirus subfamily. Further sequence comparisons of the gag gene confirmed that LPDV is most closely related to Rous sarcoma virus (RSV). However, the gag, pro, and pol open reading frames (ORFs) were in different translational phases so that the expression of their mature gene products would require the double frame-shifting mechanism utilized by simian retroviruses, mouse mammary tumor virus, and human T-cell leukemia virus. In contrast, the RSV proteinase is synthesized as part of the gag precursor. The LPDV gag gene differs from that of RSV as well as from all other retroviruses in that it encodes a unique 31,000-Da (p31) protein, located between the MA and the CA coding sequences. FOur short ORFs of unknown function were present, Whether the putative products of these ORFs account for the acute nature of LPDV-induced disease remains to be determined.

Amino Acid Sequence↗
PubMed1994

Characterization of cDNAs species encoding the Tat protein of caprine arthritis encephalitis virus.

Two distinct species of caprine arthritis encephalitis virus (CAEV) tat cDNAs were isolated early after infection of a Himalayan tahr cell line. Sequence analyses predicted that one cDNA (pCEV/e1) represented a polycistronic transcript that encodes Tat and Rev as well as an N-terminally truncated transmembrane protein and a protein, designated X, whose function is unknown; whereas the other cDNA (pCEV/f1) encodes Tat and the env gene products. pCEV/e1 trans-activated a CAEV LTR-chloramphenicol acetyltransferase reporter gene in goat synovial membrane cells. This activity was shown to be encoded by the Tat open reading frame by analysis of a deletion mutant. Because the pCAEV/f1 insert was unstable in plasmid form, its Tat activity could not be convincingly demonstrated. The target sequences for Tat within the CAEV LTR were localized to the U3 region which, when placed in either orientation upstream of heterologous promoters, was able to confer responsiveness to Tat.

Amino Acid Sequence↗
PubMed1994

Prokaryotic expression cloning of a novel human tyrosine kinase.

Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.

Amino Acid Sequence↗
PubMed1994

Diagnostic test for lymphoproliferative disease virus infection of turkeys, using the polymerase chain reaction.

Lymphoproliferative disease virus, a type-C retrovirus, is the etiologic agent of a naturally acquired lymphoproliferative disorder in turkeys. The disease is characterized by rapid induction of lymphoproliferative lesions with morphologic and histopathologic features resembling those of reticuloendotheliosis. Owing to lack of overt clinical manifestations, early detection of lymphoproliferative disease virus is essential for preventing the rapid horizontal spread of the disease that can decimate flocks. We describe development of a simple, rapid, sensitive, and highly specific assay, using the polymerase chain reaction, capable of providing differential diagnosis of the disease soon after infection.

Animals↗
PubMed1993

Fibroblast growth factor receptor 4 is a high affinity receptor for both acidic and basic fibroblast growth factor but not for keratinocyte growth factor.

A cDNA predicted to encode a transmembrane tyrosine kinase receptor with sequence features characteristic of known fibroblast growth factor (FGF) receptors was isolated from an expression library constructed from the human mammary epithelial cell line B5/589. This cDNA, designated cl44, encodes a product of 803 amino acid residues and was readily distinguishable from known FGF receptors. During the course of our studies, Partanen et al. (Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson, W. L., and Alitalo, K. (1991) EMBO J. 10, 1347-1354) isolated a new FGF receptor, designated FGFR4, from the human leukemia cell line, K562. Its amino acid sequence is identical to that of cl44 with the exception of 1 residue. The 5'-untranslated sequences of the two cDNAs diverged far upstream of the initiation codon. A myoblast line, L6E9, which lacks FGF receptors, was utilized to express high levels of FGFR4. We found, in contrast to Partenen et al., who reported only binding of acidic FGF, that FGFR4 bound both acidic and basic FGF with dissociation constants of 10-15 and 120 pM, respectively. No detectable binding of keratinocyte growth factor was observed. In studies aimed to determine whether FGF receptors contribute to the development of human tumors, we screened RNAs prepared from cell lines derived from a variety of solid tumors. High levels of the cl44 transcript were detected in 8 of 14 and 6 of 9 human mammary and kidney carcinomas, respectively, but only infrequently in other types of tumors. In contrast, FGFR1 was found to be frequently expressed in kidney, but not in breast tumor cells, suggesting a possible role for FGFR4 in human mammary cancer.

Amino Acid Sequence↗
PubMed1993

Structural and functional characterization of rev-like transcripts of equine infectious anemia virus.

Three cDNA clones representing structurally distinct transcripts were isolated from a cDNA library prepared from cells infected with equine infectious anemia virus (EIAV) by using a probe representing the S3 open reading frame, which is thought to encode Rev. One species, designated p2/2, contained four exons and was identical to a previously described polycistronic mRNA that encodes Tat. This transcript was predicted to also direct the synthesis of a truncated form of the transmembrane protein and a putative Rev protein whose N-terminal 29 amino acids, derived from env, are linked to S3 sequences. The second cDNA, p176, also consisted of four exons which were generated by two of three of the same splicing events that occur with p2/2 but not with the Tat mRNA. The alternative splice site giving rise to the second exon of p176 results in a bicistronic message that would encode the same transmembrane and Rev proteins as p2/2. The first exon of the third transcript, p20, was identical to those of p2/2 and p176 but was spliced directly to S3. This monocistronic message could encode a second form of Rev that lacks env sequences, provided that Rev synthesis would initiate at a non-AUG codon. The coding capacity of each cDNA was assessed in a eukaryotic system using S3 antisera. Two putative Rev proteins with apparent molecular masses of 18 and 16 kDa were expressed by p2/2 and p176, while p20 expressed only a 16-kDa species. Analysis of EIAV-infected cells with S3 antisera revealed the presence of an 18-kDa protein. Surprisingly, the same protein was detected in purified virions. By using a reporter construct, the chloramphenicol acetyltransferase gene linked to EIAV env sequences, we were able to demonstrate greatly enhanced chloramphenicol acetyltransferase activity in cells cotransfected with this construct and any of the three cDNAs.

Amino Acid Sequence↗
PubMed1992

The lymphoproliferative disease virus of turkeys represents a distinct class of avian type-C retrovirus.

The lymphoproliferative disease virus of turkeys (LPDV) is the etiological agent of a rapidly developing lymphoproliferative process in turkeys. To better understand the genetic relationships of LPDV to other retroviruses we determined the nucleotide sequence of its pol gene. Comparative computer analyses of the deduced amino acid sequences of the reverse transcriptase and integrase domains within pol established that LPDV represents a distinct class of avian retroviruses that is most closely related to the avian leukemia-sarcoma viruses.

Amino Acid Sequence↗
PubMed1992

Defective viral particles in caprine arthritis encephalitis virus infection.

Attempts to isolate full-length unintegrated circular forms of the caprine arthritis encephalitis virus (CAEV) genome yielded only a large number of molecules with deletions. The 3' borders of most of these deletions were near the U3 region of the long terminal repeat whereas the 5' edges were found at various upstream sites within pol or env. With one exception, gag sequences were always present. Analysis of molecular clones derived from integrated proviral CAEV genomes from the same infected cells showed a similar spectrum of deletions. The presence of transcriptionally active elements within the U3 domain of the defective genomes, as well as cis-acting elements within the leader sequences known to be required for efficient encapsidation of viral RNA, suggested that the defective viral DNA genomes could be transcribed into defective RNA molecules which could then be packaged into virions. Isopycnic density gradient centrifugation of supernatants of infected cell cultures indicated the presence of particles with densities less than that expected for intact virions (1.16 g/cc). Northern analysis revealed the presence of smaller viral-specific RNAs that lacked env sequences. These data, along with the structures of the molecular clones, suggest that CAEV stocks contain particles with defective genomes. The role of these particles in influencing the course of virus infection remains to be determined.

Animals↗
PubMed1992

The surface envelope protein gene region of equine infectious anemia virus is not an important determinant of tropism in vitro.

Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms.

Animals↗
PubMed1991

Lymphoproliferative disease virus of turkeys: sequence analysis and transcriptional activity of the long terminal repeat.

The lymphoproliferative disease virus (LPDV) is the etiological agent of a lymphoproliferative disease that naturally occurs in turkeys. Recently, we have cloned the LPDV provirus and established it as a replication-competent genome devoid of a viral oncogene [Gak et al., J. Virol. 63 (1989) 2877-2880]. This report presents the nucleotide sequence of its long terminal repeat (LTR) and establishes it as a potent transcriptional element. Several features of the LPDV LTR were similar to those found in the LTRs of the avian sarcoma-leukemia viruses (ASLV) and include the primer-binding site (tRNATrp), the polypurine tract, the organization of the polyadenylation signal, the complexities of the U3, R and U5 regions, as well as a potential secondary structure in U5-R. The LTR sequence diverges significantly from the ASLV LTRs, which share a common structure and have extensive sequence homology mainly in the R and U5 domains. These findings support the conclusion that LPDV represents a distinct class of avian retrovirus, evolutionarily related to the ASLV family.

Animals↗
PubMed1991

The Tat protein of equine infectious anemia virus is encoded by at least three types of transcripts.

Nucleotide sequence analysis of a cDNA library of EIAV-infected canine cells established a complex pattern of gene expression, characterized by alternatively spliced polycistronic transcripts. The EIAV tat gene product was shown to be encoded by at least three species of mRNA which differed in their ability to trans-activate the EIAV LTR upon expression in canine cells. The most active cDNA was monocistronic, consisting of three exons. The most abundant cDNA in the library contained four exons and was identical to a polycistronic transcript previously described (Noiman et al., 1990b) which contains open frames for Tat, putative Rev, and truncated transmembrane proteins. Products consistent in size with those predicted for these last two proteins could be detected in in vitro translation experiments. The third Tat message, another four-exon form, also potentially encodes an amino terminally truncated transmembrane protein. In vitro mutagenesis experiments and analysis of subgenomic and partial cDNA clones confirmed and extended previous findings that S1 sequences are essential for trans-activation and that Tat translation initiates at a non-AUG codon either in the full-length Tat message or in the genomic S1 open reading frame. The Tat protein (8 kDa) was detected in cells transfected with a Tat cDNA construct and in canine cells persistently infected with EIAV. The Tat activity of polycistronic mRNAs was lower than that of the monocistronic form, suggesting that the expression of the EIAV trans-activator may be subject to several levels of posttranscriptional control.

Amino Acid Sequence↗