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U Novak

Publications and source records attributed to U Novak.

50 records · Page 3Linked to original sources
PubMed1989

Molecular analysis of the human beta-globin locus activation region.

Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human epsilon-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human beta-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase beta-globin mRNA levels 8- to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed.

Animals↗
PubMed1988

Selective amplification of the cytoplasmic domain of the epidermal growth factor receptor gene in glioblastoma multiforme.

Primary brain tumors of glial origin often overexpress epidermal growth factor receptors (EGF-Rs). This may be associated with amplification of the EGF-R gene. We have examined tissue from 23 glioblastoma multiforme tumors and found amplification and rearrangement of the EGF-R gene in four of these. The cytoplasmic domain of the EGF-R gene was invariably amplified in these four tumors, while the epidermal growth factor binding domain was not uniformly amplified in three of these tumors. Western blot analysis of the EGF-R protein revealed high levels of a truncated EGF-R protein in two of the four tumors with EGF-R gene amplification.

Cytoplasm↗
PubMed1987

Heterogeneous cytogenetic abnormalities in small cell lung cancer cell lines.

Ten cell lines were established from different biopsies from nine patients with small cell lung cancer (SCLC). These were established from metastases in the marrow (7), breast (1), pleural fluid (1), and spinal cord (1) and had been in culture for periods varying from 1 to 11 months. All cell lines exhibited typical cytological features of SCLC, and produced neuron specific enolase. All lines examined (5) contained dense core granules and were tumorigenic when injected intracranially into nude mice. None of the six cell lines tested had an amplified oncogene (c-myc or n-myc) previously reported to be amplified in SCLC. Detailed chromosome analyses were undertaken and showed that a structural abnormality of chromosome 3(p11p23) was a frequent (6 of 10) but not invariable finding. Our study and one other indicate that there is not a unique chromosome abnormality present in all cases of SCLC, although loss of chromosome 13 and structural abnormalities of chromosome 3 were frequently found. A feature of these SCLC cell lines established from metastatic deposits was the complexity of the karyotypes due to numerical and structural chromosomal abnormalities.

Biopsy↗
PubMed1987

Identical mRNA for preproglucagon in pancreas and gut.

The structure of the human glucagon gene and the sequence around the putative promoter region and transcriptional start site are shown. Cloning and sequencing of the cDNAs, coding for preproglucagon from human pancreas and colon, as well as primer extension analysis of RNA from pancreas, ileum and colon point to an identical transcriptional start site in all tissues, about 3 X 10(3) base pairs upstream from the first coding exon as defined by Bell et al.

Animals↗
PubMed1984

Structure and properties of a highly repetitive DNA sequence in sheep.

Cleavage of sheep DNA with the restriction endonuclease EcoR I yields three discrete size classes (370, 435 and 800bp) of highly repetitive DNA. The 435bp long fragment was cloned and its nucleotide sequence determined. All three classes of repetitive DNA are related to each other as seen by cross-hybridisation. They are tandemly arranged in the genome and in situ hybridisation to sheep lymphocyte chromosomes show their location mainly in the centromere region of all chromosomes. The primary sequence of the repetitive DNA shows a close structural similarity to the bovine 1.715 satellite DNA, however only poor cross-hybridisation between the sheep and cattle repetitive DNA could be shown.

Animals↗
PubMed1982

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

DNA, Viral↗
PubMed1981

Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein.

A modified polyoma virus genome has been constructed which can encode the middle-T protein, but not the large-T or small-T proteins. This was achieved, starting with the full length viral DNA inserted into a plasmid vector, by replacing a small genomic restriction fragment spanning the middle-T intervening sequence with the equivalent fragment from a cloned partial cDNA copy of the middle-T protein mRNA. Transfection of the modified viral DNA into cultured rat cells efficiently induced the formation of transformed cell foci which gave rise to cell lines that grew as tumours after injection into Fisher rats. The only viral early-region antigen synthesized by the cell lines was the middle-T protein. Expression of the middle-t protein is therefore sufficient to establish and maintain a transformed state. The viral mRNA produced by two of the transformed cell lines was structurally indistinguishable from the normal middle-T mRNA found in productively infected cells, suggesting that RNA splicing is not an essential step in the biogenesis of this messenger.

Animals↗
PubMed1981

Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus.

Deletions of polyoma virus DNA around the region that codes for the C-terminus of the viral middle T-antigen were created using a transforming fragment (BamH I/EcoR I) of viral DNA cloned in the plasmid vector pAT153. These species were recloned and assayed for their ability to transform Rat-1 cells in culture. Our results showed that whereas the DNA sequence between the presumed translational termination codon for the viral middle T-antigen and the single viral EcoR I site could be removed with no apparent effect on transformation, the removal of the termination codon itself or any amino acid coding sequences of this protein caused a drastic decrease in the transforming ability of the DNA. Transfection of Rat-1 cells with plasmids that contained viral DNA with deletions which corresponded to the last fourteen or more amino acids of the middle T-antigen never gave rise to cellular transformation.

Amino Acid Sequence↗
PubMed1980

Effect of actinomycin D on nucleic acid hybridization: the cause of erroneous DNA elongation during DNA synthesis of RNA tumor viruses in vitro.

Actinomycin D, known for its suppression of cellular RNA synthesis and for the reduction of the rate of synthesis of double-stranded DNA by the RNA tumor virus RNA-dependent DNA polymerase, was found to interact with single-stranded DNA in such a way as to inhibit DNA . DNA and DNA . RNA hybridizations. This finding is discussed in the light of the observation that DNA elongation during DNA synthesis of RNA tumor viruses is blocked in vitro in the presence of actinomycin D. It thus supports the model that hybridization is a necessary step during RNA tumor virus DNA synthesis.

Avian Leukosis Virus↗
PubMed1980

Polyoma virus: some considerations on its transforming genes.

The polyoma virus genome is organized in such a way that the DNA coding capacity of the virus is maximized. Not only are there overlapping genes, but more than one reading frame within a particular region of the genome is used for coding. This is especially apparent in the 'early region', which codes for three of the known viral proteins, the large, middle and small T-antigens. Only a part of this region appears to be necessary for the transformation of cells, and it contains coding information for middle and small T-antigens, but only for a part of large T-antigen. A two-stage model for transformation is proposed which takes into account the major assays for transformation and the viral proteins that are suggested to be involved at each stage. In addition, the virus may induce a host protein to transform by an alternative route. It is suggested that the viral transforming function in polyoma virus could be of host cell origin.

Antigens, Neoplasm↗
PubMed1980

Coding capacity of a 35 percent fragment of the polyoma virus genome is sufficient to initiate and maintain cellular transformation.

Rat-1 cells were transfected with the restriction enzyme fragment of polyoma virus DNA that extends clockwise from the Bcl I site ((65.4 map units) to the EcoRI site (0/100 map units). Six transformed cell lines were obtained and one of them (BE-1) has been investigated in detail. The viral DNA that is integrated into host DNA in this line appeared to consist of two fragments arranged in a "head-to-tail" tandem with no detectable intervening host sequences. BE-1 cells contained polyoma virus small and middle tumor antigens that were indistinguishable from the corresponding tumor antigens from lytically infected cells. No large tumor antigen was detected but a "new" Mr 34,000 protein, which proved to be a truncated version of large tumor antigen, was immunoprecipitated by anti-tumor-antigen antiserum. After injection of 10(6) BE-1 cells into young syngeneic Fischer rats, tumors appeared within 3--4 weeks. Thus, the coding capacity of the Bcl I/EcoRI fragment of polyoma virus DNA is sufficient to enable the cells to produce all of small and middle tumor antigens and about a third of large tumor antigen, to transform cells stably in culture, and to produce tumors in vivo.

Animals↗
PubMed1979

Elongation of DNA complementary to the 5' end of the avian sarcoma virus genome by the virion-associated RNA-dependent DNA polymerase.

RNA-dependent DNA synthesis in a virion-associated reaction has been described as being dependent upon the detergent concentration used for disruption of the virion. In this study, the Triton X-100 concentration was found to affect the elongation of the initially synthesized DNA complementary to the last approximately 100 nucleotides at the 5' end of the RNA (cDNA100). Whereas elongation of cDNA100 increased with time of incubation at the optimal detergent concentration, this process was retarded at higher detergent concentrations. At the optimal detergent concentration, elongated DNA was of low chemical complexity, indicating that extension of cDNA100 occurred at a unique site on the RNA. Higher than optimal detergent concentrations resulted in nonspecific elongation and in DNA of high chemical complexity. This was shown by oligopyrimidine tract analysis. Furthermore, actinomycin D was observed to inhibit the elongation of cDNA100 at the optimal detergent concentration. The nature of the elongation process was elucidated by analysis of DNA synthesized in a virion-associated reaction in the presence of bacteriophage Qbeta RNA. At the optimal detergent concentration DNA complementary only to avian sarcoma virus RNA was synthesized, whereas at higher concentrations DNA was copied from both avian sarcoma virus and Qbeta RNA. We conclude that the elongation mechanism of cDNA100 is affected by the detergent concentration and elongation is unspecific at higher than optimal detergent concentrations. The mechanism by which the nonionic detergent stimulates DNA synthesis has not yet been resolve. We assume that other factors in addition to DNA polymerase are involved in elongation of cDNA100.

Alpharetrovirus↗