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Biomedical subjects

J Geryk

Publications and source records attributed to J Geryk.

At least 37 records · Page 2Linked to original sources
PubMed1992

Biology of Rous sarcoma virus adapted for replication and transformation of duck cells.

A duck-adapted variant of the Prague strain of Rous sarcoma virus and its molecularly cloned derivatives were characterized biologically. The virus replicated and transformed with the same efficiency both duck and chicken embryo fibroblasts. The daPR-RSV-C was also used for induction of tumours in newborn hamsters. The virus from virogenic hamster tumours could be rescued using the duck cells as indicator cells. The virus rescue was influenced by restricted replication of the virus in the indicator cells.

Animals↗
PubMed1991

The isopycnic, compartmentalized integration of Rous sarcoma virus sequences.

Rous sarcoma virus (RSV) can cause tumors in hamsters, which harbor complete or partially deleted RSV sequences, in their genomes. Here we have studied the localization of RSV sequences integrated into the genome of cell lines derived from six independent hamster tumors. We have found that integration occurred in the isochores richest in guanine + cytosine, of the host genome, as it had been previously observed for bovine leukemia and hepatitis B viral sequences. The integration of RSV proviral sequences is, therefore, 'isopycnic' (i.e., it takes place in host genome sequences which compositionally match the viral sequences) and compartmentalized (i.e., it occurs in a small compositional compartment of the host genome). The hamster genome compartment hosting RSV sequences precisely corresponds to a compartment of the human genome which is the most active in both transcription and recombination. The notion of a compartmentalized, isopycnic integration of RSV proviral sequences fits, therefore, with the viral integration into transcriptionally active and recombinogenic regions of the host genome observed by other authors, but is broader, in that it includes, in addition, the requirement for a compositional match between host genome sequences and expressed viral sequences.

Animals↗
PubMed1990

Ducks: a new experimental host system for studying persistent infection with avian leukaemia retroviruses.

Long-term persistence of the avian leukosis virus (ALV), the transformation-defective mutant of Prague strain Rous sarcoma virus subgroup C (td PR-C) was established in heterologous duck hosts after infection in mid-embryogenesis. Transient viraemia was observed for about 4 weeks after hatching and was lost in most of the infected ducks by about 6 months. Loss of viraemia was accompanied by the increasing synthesis of virus-neutralizing antibodies. In spite of strong virus-neutralizing antibodies, virus was detected by the cocultivation assay in duck tissues throughout the observation period up to 5 years. In the viraemic phase of infection, we found integrated proviruses in various tissues, preferentially in stomach muscle tissue and in the thymus. The long-term persistence of virus was frequently accompanied by liver necrosis and neoplastic diseases. Injection of td PR-C virus into early embryos resulted in more pronounced infection accompanied by an increased copy number of viral DNA per cell, high mortality and remarkable atrophy of thymus tissue in infected ducklings.

Animals↗
PubMed1990

A transformation-competent recombinant between v-src and Rous-associated virus RAV-1.

The LTR, v-src, LTR provirus, which arose by the reverse transcription and integration of src mRNA in the H-19 hamster tumor, has been successfully rescued by fusion with chicken fibroblasts infected with Rous-associated virus RAV-1. One rescued virus, E6, acquired 1 kilobase of the 5' end of the gag gene structure. Recombination took place in the region of 15-nucleotide homology exactly between v-src exon (position 7054) and gag (position 1417). This recombination resulted in the alteration of src splice acceptor site sequences, but this site is maintained as a functional splice acceptor site. The nucleotide structure of the long terminal repeat of recombinant E6 virus suggests that it arose by the intermolecular jump of reverse transcription from RAV-1 to src mRNA and then the switch of templates between already depicted regions of homology. The second jump of reverse transcription was apparently an intramolecular event. The acquisition of 1 kilobase of the 5' gag by E6 resulted in maintaining the balance of unspliced and spliced E6 RNAs and assured the replication advantage of rescued E6 virus over rescued F6 virus, the genome of which corresponds to that present in ancestral H-19 cells.

Animals↗
PubMed1989

Transduction of the cellular src gene and 3' adjacent sequences in avian sarcoma virus PR2257.

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.

Amino Acid Sequence↗
PubMed1989

Structural analysis of proviruses in additional hamster tumour cell lines transformed by provirus II rescued from XC cells and definition of a new cell line harbouring amplified proviruses.

Additional 18 Syrian hamster tumour cell lines induced independently with the virus rescued from XC cells (provirus type II) have been analysed by restriction mapping. Taking into account the already published results of an analysis of other six tumour cell lines, altogether 24 tumour cell lines were characterized. In 62.5% of these lines a complete proviral unit has been integrated; 20.8% of the lines harbour altered proviral structures where the 3' end of the gag gene and the entire pol gene had been deleted; 8.3% of the lines carry both such a deleted provirus and a complete provirus. Apart from the already published cryptic provirus found in H-19 cells, no such provirus has been found in another cell line. Therefore the appearance of the cryptic provirus represents an infrequent event (4.16%). Similarly, the significant amplification of proviruses accompanied with a rearrangement in the env gene was found only in one tumour cell line. H-42, in the genome of which five proviral units are accommodated. The probable arrangement of amplified proviruses and the mechanism of their genesis are discussed.

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PubMed1988

Genome structure of a new avian sarcoma virus PR2257 determined by restriction analysis.

The genome structure of a new avian src-containing acute transforming virus, designated PR2257, was studied by restriction analysis. PR2257 was isolated from a sarcoma grown in a 155-day-old chick inoculated intraembryonally with a transformation-defective mutant of daPR-C retaining no src sequences. The 4.4-kb genome of PR2257 consists of the following regions: 5' LTR--spliced version of chicken c-src--incomplete env gene--3' LTR. This structure clearly indicates that PR2257 was generated by recombination between a td daPR-C and chicken c-src.

Animals↗
PubMed1987

Restriction analysis of additional "non-producing" cell clones obtained after transformation of Japanese quail fibroblasts with viruses rescued from cryptovirogenic H-19 cells.

The proviral structure of two transforming viruses rescued from cryptovirogenic H-19 hamster cells harbouring the LTR, v-src, LTR proviral structure was analysed by restriction mapping. It was established that the rescued G8 virus keeps the same restriction pattern as that observed in the cryptic H-19 provirus and that it is integrated into the cell genome at a different position characteristic of each "non-producing" (NP) cell clone. In these features it corresponds to the previously described F6 rescued virus. In studying the E6 rescued virus we found again that it is integrated at a position characteristic of the NP cell line studied. Using a series of restriction enzymes it was specified that the E6 proviral unit acquired at least 0.25 kb from the left part of the gag gene and 0.85 kb of an unidentified DNA. Because this DNA structure was not digested with the enzymes employed that cut in RSV provirus and did not hybridize with the viral gene probes, it might be of cellular origin. The recombinational events involved in E6 virus genesis, the nature of an additional DNA structure, and the functional significance of the acquired part of the gag gene are discussed.

Animals↗
PubMed1987

Haematological and pathomorphological studies on chickens inoculated with transformation-defective sarcoma virus.

A transformation-defective mutant of Rous sarcoma virus (td PR-RSV-C) inoculated into chicken embryos caused a number of pathological changes in chickens after hatching. Disturbances in haematopoiesis were manifested by anaemia of different degree and by the presence of immature blood elements in the peripheral blood. The haematological picture of erythroblastosis was confirmed histologically. In addition to erythroblastosis, lymphoid leukosis and a proliferative lymphosarcoma-like tumour were demonstrated histologically.

Animals↗
PubMed1986

Transmission of (LTR, v-src, LTR) without recombination with a helper virus.

We have analyzed transmission of (LTR, v-src, LTR) cryptic structure integrated in the H-19 mammalian tumor cell line. From this cell line different isolates of transforming virus were rescued in heterokaryons produced by fusion with chicken fibroblasts infected by replication-competent avian leukosis virus RAV-1. One of them (F6) was used for the transformation of avian cells in the absence of the helper virus. In four transformed cell lines studied, the (LTR, v-src, LTR) structure was again integrated at a unique position in the cell DNA of each line. This indicated that the (LTR, v-src, LTR) structure is transmitted by the helper virus without recombination. This point has been further supported by the finding that a src-containing species corresponding in size to the nonpolyadenylated src mRNA is present in the RNA isolated from the rescued F6 transforming virus which might serve as template for the synthesis of (LTR, v-src, LTR) structure by the reverse transcriptase provided by RAV-1.

Animals↗
PubMed1986

Characterization of transforming viruses rescued from a hamster tumour cell line harbouring the v-src gene flanked by long terminal repeats.

The organization of proviruses derived from infecting transforming viruses rescued from hamster tumour cells was studied. Southern blot analysis indicated that the provirus from the F6 cell line was organized as long terminal repeat (LTR)-src-LTR, and S1 mapping experiments suggested that it was probably derived by reverse transcription of src mRNA followed by integration. In the E6 cell line, the provirus unit was arranged as LTR-delta gag-src-LTR, indicating a recombination event between the rescued transforming virus and the helper virus. These results suggest that transforming defective viruses containing only the src gene can be rescued from nonpermissive mammalian cells.

Animals↗
PubMed1985

Isolation of two transforming viruses from sarcomas obtained in chickens inoculated intraembryonally with a transformation defective mutant of Prague strain Rous sarcoma virus.

Sarcomas appeared after long latency with a frequency of about 2% in Brown Leghorn and (CB X IC)F1 chickens after intraembryonic and neonatal inoculation of transformation-defective mutants of ASVs subgroup C. Only the freshly isolated td mutants, td daPR-C and td daPR-C morphf, exhibited the tumorigenic activity, whereas the standard td mutants induced no sarcomas. Two (862 and 2257) out of four tumours could be transplanted in young chickens and produced low titres of transforming virus. The other two tumours were not transplantable and devoid of any transforming virus. Viruses 862 and 2257 are clearly defective in replication, virus 2257 cannot be complemented efficiently by any helper virus in vitro. The low titre of virus 2257 is not caused by interference with a helper virus of the same subgroup specificity. Both viruses are highly tumorigenic in vivo.

Animals↗
PubMed1985

Identification of transformation-specific proteins synthesized in cryptovirogenic mammalian cells.

The cryptovirogenic PR-RSH-19 (H-19) hamster cell line, derived originally from a tumour induced by the virus rescued from XC cells by transfection, was studied. The pp60v-src protein was detected by immunoprecipitation with sera from tumour-bearing rabbits (TBR sera) from lysates of H-19 cells. A closely related protein p60 was precipitated by antiserum obtained from hamsters with tumours induced by H-19 cells. The pp60v-src of H-19 cells possesses an associated kinase activity. These results are consistent with the notion that the detected pp60v-src protein is a product of the cryptic sequences in H-19 cells.

Animals↗
PubMed1984

Characterization of cryptovirogenic, virus-productive and helper-dependent virogenic hamster tumour cell lines.

Three hamster cell lines were established from tumours induced with the virus rescued from XC cells. The H-20 cell line produces little infectious transforming virus and synthesizes uncleaved Pr76 (product of gag gene) in the amount comparable to that obtained from PR-RSV-C-infected permissive cells. Like the helper-dependent virogenic H-18 cell line, it however produces only a marginal amount of viral glycoprotein (env gene product) as revealed by the complementation assay. Even a lower amount of the env gene product almost escaping detection by the complementation test has been found in the helper-dependent virogenic H-18 cell line. This cell line has also been shown to produce a low quantity of the gag gene product. The H-19 cell line synthesizes no detectable gag or env gene products and is not inducible by cell fusion with chicken fibroblasts even when preinfected with the replication competent td PR-C. This is in agreement with data from restriction analysis (Svoboda et al. 1983) showing that H-19 cells contain only cryptic proviral sequences represented by v-src and LTR. The three cell lines together with those described previously (Svoboda 1981) were analysed karyologically. Random changes in chromosome numbers and structural alterations in individual chromosomes were only found.

Animals↗
PubMed1983

Characterization of exogenous proviral sequences in hamster tumor cell lines transformed by Rous sarcoma virus rescued from XC cells.

Alterations in viral structural genes have been studied in five cell lines derived from Syrian hamster tumors which had been induced by the virus rescued from XC cells by transfection. Two cell lines, H-18 and H-20, have all the viral structural genes expressed, but a new EcoRI recognition site appeared in the region of the pol gene sequence. Provirus present in H-12 lacks the 3' part of the gag gene sequences as well as the pol gene, therefore, it gives rise to an anomalous 1.8 Md EcoRI fragment. This line also does not synthesize viral RNA of genomic size, and none of the subgenomic RNAs found hybridized with the DNApol probe. The H-19 cell line harbors only the src gene and LTR sequences, the U3 part of which seems incomplete or different from that of PR-RSV. The cryptic proviral structure in H-19 is transcribed into src mRNA. The degree of transcription of the src gene is about 25 viral RNA equivalents per cell. The H-9 cells harbor the complete provirus and, in addition, proviral structures having the deletion in gag-pol genes. The possible ways of development of provirus alterations and the role of cryptic proviral sequences in oncogenesis are discussed.

Animals↗
PubMed1982

Characterization of a mutant of the Prague strain of Rous sarcoma virus inducing fusiform transformation of avian fibroblasts in vitro.

The DNA as isolated from duck fibroblasts transformed by a duck-adapted Prague strain of Rous sarcoma virus and used for transfection. Transformed recipient BLEF and DEF cultures exhibited considerable morphological variability. The virus designated daPR-RSV-C morphf was obtained from the culture with fusiform transformation and cloned. The virus retained the ability to induce fusiform transformation, even after 20 passages on chicken fibroblasts. There was a good correlation between focus forming activity of the virus and its tumorigenicity in chickens. The frequency of morphf mutation to another phenotype was less than 10(-3) in cloned virus. Foreign avian embryonic cells transformed by this virus clone had a similar morphological appearance as transformed chicken cells. The clone also retained two additional non-conditional markers - subgroup C specificity and the ability to replicate efficiently in duck cells ("duck adaptation"). Freshly obtained cloned virus was found not to contain a transformation-defective mutant. Such a mutant occurred in the second passage of the virus of DEF where the mutant was isolated. Inoculation of the td mutant into Brown Leghorn embryos gave rise to a sarcoma in one of the 36 examined chickens. However, no transforming virus was detected in the sarcoma. SDS-polyacrylamide gel electrophoresis showed that cloned daPR-RSV-C morphf contained only genomic RNA; its molecular weight 3.08 X 10(6) daltons corresponded to the molecular weight of a non-defective PR-RSV-C used as control.

Animals↗
PubMed1980

Replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus and isolation of a td mutant from duck-adapted PR-RSV-C.

The replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus subgroup C was studied using roller cultures. Under such conditions, 10(5)--10(6) infectous units of virus per 0.2 ml were produced, as revealed in both the reverse transcriptase and 16Q complementation tests. A new td daPR-RSV-C mutant was isolated from duck-adapted PR-RSV-C. This mutant replicated in roller cultures with equal efficiency as the original td PR-RSV-C. It was verified that td daPR-RSV-C does not transform chicken fibroblasts, is not oncogenic for 3-week-old chickens and has subgroup C host-range specificity. Both td mutants replicate in duck cells and reach the same titres.

Animals↗