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Biomedical subjects

F O Wettstein

Publications and source records attributed to F O Wettstein.

At least 37 records · Page 2Linked to original sources
PubMed1989

Genetic analysis of CRPV pathogenesis: the L1 open reading frame is dispensable for cellular transformation but is required for papilloma formation.

Genetic studies to elucidate the role of papillomaviruses in the development and progression of tumors have been severely hampered because the viruses cannot be grown in tissue culture and therefore mutants are not available. We have employed recombinant DNA for papilloma induction to identify essential sequences involved in papillomavirus pathogenesis. Here, we demonstrated that deleting most of the open reading frame (ORF) L2 did not affect the potential of viral cottontail rabbit papillomavirus (CRPV) DNA to induce papillomas. The extrachromosomally maintained DNA in the papillomas was not rearranged and the major transcripts of 1.3 and 2.0 kb encoded E7 and E6, respectively. A recombinant DNA containing a larger deletion lacking the 3' terminal half of ORF L2 and all of ORF L1 (pdlBc/l) did not induce papillomas. The results indicate that sequences in the late region not required for transformation of NIH 3T3 cells by bovine papillomavirus type-1 essential in CRPV for induction of papillomas.

Animals↗
PubMed1989

Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms.

A new promoter located within E6 was mapped in human papillomavirus type 6b (HPV6b)- and HPV11-containing benign genital condylomata (genital warts). The RNA transcribed from this promoter represented the major RNA species colinear with open reading frames E6 and E7 and can encode the E7 protein. No equivalent promoter was active in HPV16-containing cancers and cancer-derived cell lines. In those, the major transcripts contained one of two different introns within E6 and the RNAs could encode two different E6 proteins and E7.

Amino Acid Sequence↗
PubMed1988

A study of pituitary thyrotropin, its subunits, and messenger ribonucleic acids in nonthyroidal illness.

We studied serum TSH, pituitary TSH and alpha and beta-subunits of TSH and their messenger ribonucleic acids (mRNAs) in three models of nonthyroidal illness (NTI) in the rat, ie diabetes mellitus (1 wk after 65 micrograms streptozotocin/g BW IP), turpentine oil-injection (8 to 48 hours after after a dose of 5 microliter/g bw SC), and complete fasting for 72 hours. Euthyroid, hypothyroid (two months after thyroidectomy) and hyperthyroid rats (30 micrograms T4/d X 7, SC) were also studied for comparison. Pituitary TSH, alpha and beta subunits and serum TSH, T4, and T3 were measured by RIA. Pituitary mRNAs coding for common delta and TSH-beta subunits were determined by cytoplasmic dot hybridization technique using specific [32P]-cDNA probes. In all NTI models there were significant decreases in serum levels of TSH, T4, and T3, but no significant changes were observed in the pituitary content of TSH, and alpha and TSH-beta subunits. Hypothyroid rats had an increase in serum TSH, pituitary TSH, and pituitary TSH-beta subunit and a decrease in pituitary alpha subunit. On the other hand, hyperthyroid rats showed a decrease in serum TSH, pituitary TSH, and pituitary TSH-beta subunit, while there was no change in the alpha subunit. A significant reduction in the pituitary TSH-beta mRNA levels was observed in all NTI models and hyperthyroidism, while TSH-beta mRNA was increased in thyroidectomized rats. alpha-mRNA was increased only in the pituitary of hypothyroid rats; there was no appreciable change in the pituitary alpha-mRNA in the various other pituitary groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Identification and characterization of the CRPV E7 protein expressed in COS-7 cells.

The papillomavirus E7 protein may play an important role in oncogenesis as it is the major viral protein present in human cervical cancer-derived cell lines. Because of the relevance of the cottontail rabbit papillomavirus (CRPV) system for the study of viral-induced malignancies, we characterized its E7 protein expressed by a heterologous strong promoter. An E7-specific antiserum was obtained by immunizing rabbits with a Trp E-E7 fusion protein containing the 88 carboxyl-terminal amino acids of E7. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS-7 cells a 14-kDa protein. The protein was present only in the soluble cytoplasmic fraction and exhibited a heterogeneous sedimentation rate in nondenaturing glycerol gradients.

Animals↗
PubMed1988

The two proteins encoded by the cottontail rabbit papillomavirus E6 open reading frame differ with respect to localization and phosphorylation.

Cottontail rabbit papillomavirus-induced tumors contain two E6-coding transcripts. A major transcript can code for a short E6 protein initiated at AUG codon 2, and a minor one could code for a long E6 initiated at AUG 1. We have identified the two proteins expressed in COS-7 cells (M. Barbosa and F. O. Wettstein, J. Virol. 61:2938-2942, 1987). The properties of the two proteins are distinctly different. The long E6 is predominantly present in the nucleus, in which it appears to be associated with the nuclear matrix. Minor portions of the long E6 are located in equal amounts in both the soluble cytoplasmic and the membrane fractions. The short E6 is a soluble cytoplasmic protein phosphorylated at serine residues.

Animals↗
PubMed1988

E2 of cottontail rabbit papillomavirus is a nuclear phosphoprotein translated from an mRNA encoding multiple open reading frames.

The papillomavirus E2 protein is a transcription trans-activator and as such as a paramount effect on viral functions. We have identified and characterized the cottontail rabbit papillomavirus E2 protein expressed from the late simian virus 40 promoter in COS-7 cells. E2 was shown to be a highly phosphorylated 49-kilodalton protein. Subcellular fractionation and indirect immunofluorescent staining indicated that E2 was located in the nuclei. E2 was expressed from a vector which contained just the open reading frame. ORF E2 and also from vectors which extended farther upstream and also expressed E7 or the short E6 protein. However, the level of E2 was very low in cells transfected with a vector expressing the long E6 protein. Mapping of transcripts with nuclease S1 and exonuclease ExoVII in cells expressing the short E6 and E2 proteins showed that E2 was translated from an RNA which encoded the short E6, E7, and E2 proteins but served as mRNA for only the short E6 and E2 proteins.

Animals↗
PubMed1987

A variant of CRPV DNA preferentially maintained as a plasmid in NIH 3T3 cells and characterization of its transcripts in nude mouse tumors.

Rabbit and human papillomaviruses are strictly epitheliotropic and their DNA replicates extrachromosomally in benign lesions (warts) of their natural host. Their tissue and host specificity is thought to be genetically controlled and may account for the inefficiency in transforming heterologous cells. In our hands, CRPV DNA did not induce foci in NIH 3T3 cells and in cotransfections with a selectable marker only integrated DNA was found. The viral DNA appeared to be transcriptionally inactive since no transcripts could be detected and in cells were not tumorigenic for nude mice. In contrast to these results a spontaneously derived CRPV variant DNA was able to replicate extrachromosomally and a majority of focus-derived cell lines were tumorigenic for nude mice. In nude mouse tumors the variant DNA remained exclusively extrachromosomal and viral transcripts were detected. The sizes of the major transcripts were 2.0 and 1.3 kb and this suggested that E6 and E7 were expressed. The situation in rabbit tissue was different; both variant and wild-type DNA were maintained extrachromosomally. The extrachromosomal maintenance of the variant but not of wild-type DNA in mouse cells suggests that the variant lacks sequences which may play a role in the host and the tissue restriction of CRPV. The deletion in the variant DNA was located in the late region and included most or all of L1 and a carboxy terminal segment of L2. A second deletion eliminated some pBR322 sequences.

Animals↗
PubMed1987

Identification of the major cottontail rabbit papillomavirus late RNA cap site and mapping and quantitation of an E2 and minor E6 coding mRNA in papillomas and carcinomas.

The capsite of the 2.6- and 4.8-kb major late transcripts of cottontail rabbit papillomavirus (CRPV) has been mapped by primer extension. A leader exon of about 300 nucleotides common to both RNAs is located in the untranslated region of the genome upstream of the capsites for early transcripts. In contrast to the early capsites which are all preceded by TATA boxes, no such sequence is present 30 nucleotides upstream of the late capsite. These data indicate that the switch from early to late transcription involves recognition of a new promoter and suppression of transcription termination at the early polyadenylation site. We have also identified a minor exon with a coding potential for a putative E2 transactivating protein. Quantitation by S1 mapping of the E2 coding exon and a minor exon coding for a full-sized E6 protein unique in size to the highly oncogenic CRPV did not reveal differences in the level of transcription between papillomas and carcinomas.

Animals↗
PubMed1987

The major human papillomavirus protein in cervical cancers is a cytoplasmic phosphoprotein.

In a previous study, the most abundant viral transcript in a human papillomavirus type 16-associated cervical cancer and in a cancer-derived cell line was characterized, and its translation product, the E7 protein, was identified (D. Smotkin and F. O. Wettstein, Proc. Natl. Acad. Sci. USA 68:4680-4684, 1986). Here we show that the E7 protein had a half life of about 1 h and was located in the soluble cytoplasmic fraction. The protein was phosphorylated at serine residues and exhibited a high heterogeneous sedimentation rate in nondenaturing glycerol gradients, suggesting an oligomer formation or association with cellular protein.

Cell Line↗
PubMed1987

Transcription of the cottontail rabbit papillomavirus early region and identification of two E6 polypeptides in COS-7 cells.

Cottontail rabbit papillomavirus (CRPV) early proteins are present at very low levels in virus-induced tumors and cannot be detected by immunological methods. Furthermore, cells in culture are not readily transformed by the virus. To overcome these difficulties in identifying and characterizing the putative transforming protein(s) coded by the E6 open reading frame, the early cottontail rabbit papillomavirus region was expressed under the control of the late simian virus 40 promoter. Mapping of the transcripts in transiently transfected COS-7 cells indicated that transcription was initiated in the late region of simian virus 40. Two E6-coded polypeptides were identified, representing translation products initiated at the first and second AUG codons.

Animals↗
PubMed1986

Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein.

Human papillomavirus type 16 DNA and RNA were characterized in the cervical cancer-derived CaSki cell line, which contains only integrated DNA, and in a cervical cancer, which contains predominantly plasmid DNA. In both, a major RNA can code for the early open reading frame E7 and a minor one can code for E6. The cervical cancer, but not the CaSki cell line, contains a minor RNA that can code for an intact E2 protein, and this may relate to the continued presence of plasmid DNA. The RNA mapping data suggest that the poly(A)+ RNA is transcribed from a minor fraction of the several hundred gene copies present, and in the cervical cancer these genomes appear to be integrated. The E7 protein has been identified in CaSki cells and the prevalence of its mRNA suggests a possible function in progression to, or long-term maintenance of, the malignant state.

Cell Line↗
PubMed1986

Human papillomavirus deoxyribonucleic acid in adenocarcinoma and adenosquamous carcinoma of the uterine cervix.

This report describes the detection of human papillomavirus type 16 or 18 deoxyribonucleic acid (DNA) in nine of 15 invasive tumors of the cervix, including three squamous carcinomas, four adenosquamous carcinomas, one glassy cell carcinoma, and one adenocarcinoma. The viral DNA was identified by Southern blotting and DNA hybridization. Human papillomaviruses may play an etiologic role in the development of at least some adenocarcinomas and adenosquamous carcinomas as well as most squamous tumors of the cervix.

Adenocarcinoma↗
PubMed1984

Cottontail rabbit papillomavirus-specific transcripts in transplantable tumors with integrated DNA.

The viral DNA and viral transcripts of a VX2 and a VX7 transplantable carcinoma lines were analyzed. Both tumor lines contain equivalent numbers of gene copies per cell of exclusively integrated viral DNA. Integration is in head-to-tail tandem repeats, in probably three sites and at least two integration sites are common. In both tumors, about half of the viral genomes share a 90-bp deletion. Common integration sites and deletions suggest that the two tumor lines were derived from the same parent tumor. The tumors contain the same major viral transcripts of 1.3 and 2.0 kb also found in both papillomas and primary carcinomas of domestic rabbits [M. Nasseri, F. O. Wettstein, and J. G. Stevens, J. Virol. 44, 263-268 (1982)]. However, the carcinoma lines differ in their minor transcripts. VX7 has four minor transcripts of 5.2, 3.8, 3.6, and 3.0 kb and VX2 has two of 4.8 and 3.0 kb. The 4.8-kb transcript is relatively major and appears to be identical to a major transcript present in virus-producing tumors of cottontail rabbits [M. Nasseri and F. O. Wettstein, J. Virol. 51, 706-712 (1984)].

Animals↗
PubMed1984

Differences exist between viral transcripts in cottontail rabbit papillomavirus-induced benign and malignant tumors as well as non-virus-producing and virus-producing tumors.

Five major cottontail rabbit papillomavirus-specific polyadenylated RNA species with sizes of 4.8, 2.6, 2.0, 1.3, and 0.9 kilobases (kb) were found in virus-producing tumors of cottontail rabbits (the natural host for the virus). Two of the RNA species (sizes, 2.0 and 1.3 kb) are indistinguishable with respect to size and map position from the RNA species detected previously in non-virus-producing benign and malignant tumors (Nasseri et al., J. Virol. 44:263-268, 1982). The 2.0-kb RNA in virus-producing benign tumors is more abundant than the 1.3-kb RNA. This, together with similar observations of benign non-virus-producing tumors, suggests that the predominance of the 2.0-kb RNA is a general feature of benign tumors. The change to a preferential synthesis of the 1.3-kb RNA appears to be a phenomenon of tumor progression from papillomas to carcinomas. Three transcripts of 4.8, 2.6, and 0.9 kb are unique to virus-producing tumors. The RNA molecules were mapped in two steps. First, hybridization of Northern blots with subgenomic probes revealed the approximate map position of the transcripts. Second, with nuclease S1 and exonuclease VII mapping procedures and end-labeled probes, the major exons of the 4.8-, 2.6-, 2.0-, and 1.3-kb RNAs were mapped precisely, and it is shown that all RNAs are transcribed from the same DNA strand. Both 1.3- and 2.0- kb RNAs consist of two exons which are separated by an identical 2.45-kb intron. The 5' ends of the 5'-proximal exons of the 2.0- and 1.3-kb RNAs map to positions 0.07 and 0.16, respectively. Some of the 2.0-kb RNA molecules, especially in the carcinoma, have an alternative 5' end at position 0.06. The 3' ends of both exons map to position 0.22, where two ends were found about seven nucleotides apart. The sizes of the 5'-proximal exons of the 2.0- and 1.3-kb RNAs are 1.23 and 0.48 kb, respectively. The 1.3- and 2.0-kb RNAs share a common 3'-proximal exon of 0.66 (0.61) kb. This exon has two 5' ends 50 nucleotides apart at map position 0.53 and a 3' end at map position 0.61. Only the 3'-proximal part of the 4.8- and 2.6-kb RNAs have been mapped precisely. Both RNAs share a common 3' end at position 0.99. The 2.6-kb RNA part consists of a single 1.59-kb exon which extends to map position 0.79.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Shope papilloma virus DNA is extensively methylated in non-virus-producing neoplasms.

The extent of viral DNA methylation in non-virus-producing benign and malignant tumors induced by the Shope (rabbit) papilloma virus was investigated with the two isoschizomeric restriction endonucleases MspI and HpaII. The overall extent of methylation is variable in papillomas and uniformly higher in carcinomas. In a transplantable Shope virus-induced carcinoma and in two transplantable carcinoma derived cell lines, the viral DNA is even more highly methylated than in carcinomas induced by the virus directly. However, even in these cells at least two MspI sites remain essentially unmethylated and these sites are also unmethylated in carcinomas induced by the virus directly. At least one additional MspI site is unmethylated in papillomas but is partly methylated in carcinomas. Viral DNA from papillomas, which consists mostly of unit-sized form I and form II DNA and from carcinomas in which large oligomeric forms predominate was separated into fractions containing viral plasmids of different size and conformation. The analysis of the different fractions showed that methylation patterns do not correlate with the size of viral DNA, but they rather correlate with the degree of neoplasia.

Animals↗
PubMed1982

Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.

The state of rabbit (Shope) papilloma virus DNA in virus-induced nonproducing tumors on domestic rabbits was investigated. Virus-specific sequences were resolved into many distinct bands by one-dimensional agarose gel electrophoresis. Two-dimensional gel electrophoresis, CsCl/propidium iodide density equilibrium centrifugation, partial digestion with a restriction endonuclease, and S1 nuclease digestion permitted us to identify the bands as free viral episomes representing circular molecules of increasing size. In some tumors (both papillomas and carcinomas), up to 25% of the virus-specific DNA was linear and comigrated with cellular DNA. Integration of at least some of these sequences was suggested by the detection of viral-cellular junction bands in one tumor after digestion of DNA with EcoRI and Sal I, enzymes that cut Shope DNA once. Finally, the physical states of viral DNA in papillomas and carcinomas were found to be similar, although free episomes were generally larger in carcinomas.

Animals↗
PubMed1982

Two colinear and spliced viral transcripts are present in non-virus-producing benign and malignant neoplasms induced by the shope (rabbit) papilloma virus.

The nature of Shope virus-specific RNA was investigated in non-virus-producing Shope (rabbit) papilloma virus-induced benign and malignant domestic rabbit tumors and in a cell line derived from the VX-7 transplantable carcinoma. RNA transfer (Northern) blot analysis of polyadenylated RNA isolated from whole cell extracts of all three sources was resolved into two major bands of 1.3 and 2.0 kilobases. Two additional minor bands of 3.5 and 4.8 kilobases could be seen in some analyses. RNA of the VX-7 cell line was further separated into nuclear, cytoplasmic, and polysomal fractions. The cytoplasmic fraction only contained the 1.3- and 2.0-kilobase species, and virus-specific RNA was found to be associated with polysomes. The two major transcripts present in VX-7 cells were mapped by hybridization of RNA transfer blots with subgenomic probes, and the results indicated that both transcripts are spliced and are most likely colinear. Our results are consistent with the suggestion that the two viral transcripts are necessary for induction and maintenance of the neoplasms.

Animals↗