PubMed Health⌕ Search

Biomedical subjects

P Sutrave

Publications and source records attributed to P Sutrave.

At least 19 recordsLinked to original sources
PubMed2000

The induction of skeletal muscle hypertrophy by a ski transgene is promoter-dependent.

The chicken c-ski gene expresses at least three alternatively spliced messages. Transgenic mice expressing proteins from cDNA corresponding to two of these messages (FB27 and FB29) under the control of a murine sarcoma virus (MSV) long terminal repeat (LTR) express the transgene in skeletal muscle and develop a muscular phenotype. Both a biologically active form of c-ski and the MSV LTR are required for the development of the muscular phenotype. The normal c-ski gene linked to two other tissue-specific promoters failed to induce muscle growth in transgenic mice, as did an inactive mutant of c-ski expressed under the control of the MSV LTR.

Animals↗
PubMed1999

Organization and chromosomal mapping of mouse Gh/tissue transglutaminase gene (Tgm2).

The mouse Gh/tissue transglutaminase gene (Tgm2), coding a dual-function protein that both binds guanosine triphosphate (GTP) and catalyzes the posttranslational modification of proteins by transamidation of glutamine residues, has been cloned. Sequence analysis of Tgm2 and comparison with the TGase sequences of other species allowed correction of several apparent sequencing artifacts in the Tgm2 cDNA. Tgm2 spans approximately 34 kb and has 13 exons and 12 introns. Although the structure of Tgm2 shows similarity to that of other transglutaminase genes, with introns ranging from 921 bp to >5 kb, several introns differ considerably in size from those of the human Gh gene, TGM2. Tgm2 maps to the distal region of mouse chromosome 2, a region syntenic to human chromosome 20q containing TGM2. Tgm2 is in the vicinity of two uncloned mouse mutations, diminutive (dm) and blind-sterile (bs). Genomic DNA from dm mice was unavailable; however, Southern blot analysis of bs DNA showed no gross rearrangements of Tgm2.

Amino Acid Sequence↗
PubMed1999

Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model.

Insulin-like growth factor-I (IGF-I) is an important autocrine/paracrine mediator of skeletal-muscle growth and development. To develop a definitive cultured cell model of skeletal-muscle hypertrophy, C2C12 cells were stably transfected with IGF-I and clonal lines developed and evaluated. Quantitative morphometric analysis showed that IGF-I-transfected myotubes had a larger area (2381+/-60 micrometer2 versus 1429+/-39 micrometer2; P<0.0001) and a greater maximum width (21.4+/-0.6 micrometer versus 13.9+/-0.3 micrometer; P<0.0001) than control C2C12 myotubes, independent of the number of cell nuclei per myotube. IGF-I-transfected myotubes had higher levels of protein synthesis but no difference in DNA synthesis when compared with control myotubes, indicating the development of hypertrophy rather than hyperplasia. Both lactate dehydrogenase and alanine aminotransferase activities were increased (3- and 5-fold respectively), and total lactate levels were higher (2.3-fold) in IGF-I-transfected compared with control myotubes, indicating an increase in anaerobic glycolysis in the hypertrophied myotubes. However, expression of genes involved in skeletal-muscle growth or hypertrophy in vivo, e.g. myocyte nuclear factor and myostatin, was not altered in the IGF-I myotubes. Finally, myotube hypertrophy could also be induced by treatment of C2C12 cells with recombinant IGF-I or by growing C2C12 cells in conditioned media from IGF-I-transfected cells. This quantitative model should be uniquely useful for elucidating the molecular mechanisms of skeletal-muscle hypertrophy.

Animals↗
PubMed1999

Association of specific DNA binding and transcriptional repression with the transforming and myogenic activities of c-Ski.

The ski oncogene encodes a transcription factor that induces both transformation and muscle differentiation in avian fibroblasts. The first 304 amino acids of chicken Ski, the transformation domain, are both necessary and sufficient to mediate these biological activities. Ski's biological duality is mirrored by its transcriptional activities: it coactivates or corepresses transcription depending on its interactions with other transcription factors. Ski represses transcription through specific binding to GTCTAGAC (GTCT element) but it possesses a transferable repression activity that can function independently of this DNA element. In this study, we locate this repression domain to the NH2-terminal two-thirds and the GTCT binding region to the COOH-terminal one-third of Ski's transformation domain. Mutations in the transformation domain of c-Ski reveal a strong correlation between GTCT-mediated transcriptional repression and the biological activities of transformation and myogenesis. We also show that a dimerization domain located at the COOH terminal end of the Ski protein increases its transforming activity and its binding to GTCTAGAC.

Animals↗
PubMed1995

Activation of a muscle-specific enhancer by the Ski proto-oncogene.

In transgenic mice, muscle-specific expression of the c-ski oncogene induces hypertrophy exclusively in a subset of fast muscle fibers. Here we report that regulatory elements from two genes expressed in fast fibers, myosin light chain 1/3 (MLC) and muscle creatine kinase (MCK), were activated when co-transfected with c-ski expression vectors in myoblasts. The expression from the MLC enhancer was reduced when the c-ski oncogene was cotransfected with MyoD into NIH3T3 fibroblasts. Activation of the MLC enhancer by Ski also occurred in vivo, since bigenic progeny generated by mating MLC-CAT and MSV-skitransgenic mice displayed higher CAT activity in their muscles than did the MLC-CAT parental line. Identification of gene targets for the fiber-specific action of the c-ski gene product provides a molecular model that could be used for the further dissection of Ski-induced hypertrophy, both in tissue culture and in vivo.

3T3 Cells↗
PubMed1995

Regulation of c-ski transgene expression in developing and mature mice.

The control of c-ski transgene expression and muscle hypertrophy have been investigated in transgenic mice. In adult animals, the level of transgene expression is linked to the specialized phenotype of individual muscles, high levels occur in fast muscles and significantly lower levels in muscles with high metabolic activity (diaphragm, soleus). These findings have led us to propose that a threshold must be passed before ski-induced growth can occur. We now show that within fast muscles, induced hypertrophy uniquely involves IIb fibers. This pattern of expression is under development control; levels of c-ski mRNA are low in all muscles at birth. In the diaphragm, there is a sevenfold increase in c-ski message levels between 5 d and maturity. By contrast, in fast extensor digitorum longus and anterior tibial muscles, there is a 24-fold increase in levels between 5 and 12 d postpartum. Muscle hypertrophy and antibody staining for c-ski protein in myofiber nuclei emerge concurrently. This pattern of c-ski expression parallels the appearance of IIb myosin heavy chain transcripts (Wydert et al., 1987) and differentiation of IIb fibers, suggesting that amplification of c-ski mRNA levels is linked to the development of IIb fiber specialization. Manipulations that are known to perturb IIb fiber development, neonatal denervation, and neonatally induced hypothyroidism inhibit high levels of c-ski expression and hypertrophy. In the adult fast EDL, denervation leads to rapid atrophy of IIb fibers and a significant decline in levels of c-ski mRNA. The results suggest that the environment of differentiated IIb fibers permits the expression of high levels of c-ski mRNA and this, in turn, induces hypertrophy.

Aging↗
PubMed1994

Isolation, identification and characterization of the FUN12 gene of Saccharomyces cerevisiae.

We have cloned and characterized the FUN12 gene which is found on chromosome 1 of Saccharomyces cerevisiae. The complete nucleotide (nt) sequence of the cDNA and the genomic clones shows that FUN12 is expressed as a 3.7-kb message and should encode a 97 kDa-protein. Immunoprecipitations using antipeptide antibodies showed that the cells contain a Fun12p of this size. The databases contain no nt sequences that are homologous to FUN12 and no protein homologous to Fun12p. Gene disruption experiments showed that FUN12 is an essential gene.

Amino Acid Sequence↗
PubMed1991

Activation of the c-ski oncogene by overexpression.

The v-ski oncogene is a truncated version of the cellular proto-oncogene, c-ski, and lacks sequences coding for both the N- and C-terminal ends of the c-ski protein. In the region of overlap, v-ski and c-ski differ by only one amino acid. To determine whether these differences underlie v-ski's oncogenic activation, we have cloned cDNAs for several alternatively spliced c-ski mRNAs and introduced these cDNAs into replication-competent retroviral vectors. The biological activities of these c-ski constructs have been compared with those of v-ski. We found that all c-ski gene products, when expressed at high levels from the promoter in the retroviral long terminal repeat, can induce morphological transformation, anchorage independence, and muscle differentiation in avian cells. Cells that are susceptible to ski-induced transformation and myogenesis normally express endogenous c-ski at low levels. Thus, it appears that overexpression of ski is sufficient for oncogenic and myogenic activation.

Animals↗
PubMed1990

ski can cause selective growth of skeletal muscle in transgenic mice.

We have created several lines of mice that contain a truncated chicken c-ski cDNA linked to an MSV LTR promoter. Adult mice from three independent lines show large increases in skeletal muscle. All three lines of mice express high levels of c-ski mRNA and protein in skeletal muscle. All other tissues examined show little or no expression of the c-ski transgene. The muscles of one of the three lines were examined in more detail. Type II fast fibers undergo selective hypertrophy in affected muscles of this line.

Animals↗
PubMed1990

Characterization of chicken c-ski oncogene products expressed by retrovirus vectors.

We constructed replication-competent avian retrovirus vectors that contain two of the three known types of chicken c-ski cDNAs and a third vector that contains a truncated c-ski cDNA. We developed antisera that recognize the c-ski proteins made by the three transforming c-ski viruses. All three proteins (apparent molecular masses, 50, 60, and 90 kilodaltons) are localized primarily in the nucleus. The proteins are differentially phosphorylated; immunofluorescence also suggests that there are differences in subnuclear localization of the c-ski proteins and that c-ski protein is associated with condensed chromatin in dividing cells.

Amino Acid Sequence↗
PubMed1990

Structure, tissue-specific expression, and transforming activity of the mouse met protooncogene.

A 6.7-kilobase met complementary DNA (cDNA) was isolated from a pcD cDNA library prepared from C3H mouse fibroblast cell line polyadenylated RNA. Sequence analysis of 6.7-kilobase met cDNA insert revealed that it contained the entire open reading frame and shared an overall homology of 88.1% with the human met gene. Using the mouse met cDNA as probe, high levels of met expression were observed in the kidney, brain, lung, skin, and embryonic tissue as well as in several factor responsive mouse myeloid cell lines. Under SV40 promoter control, the mouse met protooncogene cDNA in the pCD vector was able to transform NIH 3T3 cells. These transformed cells possess multiple copies of mouse met cDNA and exhibit properties of malignant cells, including growth in soft agar and induction of tumors in nude mice. Tumor explant cell lines analyzed by Western blot also reveal the presence of high levels of Mr 170,000 and 140,000 met protein product(s).

Amino Acid Sequence↗
PubMed1990

Vectors and genes for improvement of animal strains.

Strain improvement of agriculturally important animals will require efficient techniques for gene delivery, the ability to regulate the expression of the newly introduced genes and, most important, the identification of genes whose appropriate expression could cause improvement of the animal. We have developed a series of avian retroviral vectors that can be used to introduce new genetic information into the germ line of chickens, for which transgenics cannot be created by direct microinjection of DNA into fertilized eggs. We have identified a 220-bp segment of the chicken skeletal muscle alpha-actin gene that can cause other genes to be expressed specifically in striated muscle. This chicken promoter shows correct tissue specificity in transgenic mice and presumably could be used in other mammalian species. The skeletal muscle alpha-actin promoter has been inserted into the avian retroviral vectors and the promoter is functional in cultured cells infected by these retroviral vectors. The tissue specificity of the expression of the skeletal muscle alpha-actin promoter carried by the retroviral vectors will soon be tested in vivo. We are studying two types of genes that might be useful in strain improvement; genes that could produce dominant resistance to infection by pathogenic viruses, and genes that could play critical roles in muscle development. Expression of the envelope glycoprotein of retroviruses can specifically block the cellular receptor that viruses use to infect a susceptible cell. Expression of the avian leukosis virus subgroup A envelope in transgenic chickens prevents infection by pathogenic viruses of the same subgroup. We are attempting to block reticuloendotheliosis virus infection by expressing the reticuloendotheliosis envelope glycoprotein. We have shown that we can block infection in cultured cells, and we are now creating retroviral vectors for experiments in vivo. We have also begun to study the cellular homologue of the ski oncogene, which has been shown to stimulate the differentiation of quail myoblasts in vitro. Biologically active cDNAs have been isolated; we have now begun to analyse the effects of expressing the c-ski proteins in the whole animal.

Animals↗
PubMed1987

Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors.

We have previously described several helper independent vector constructions (S. Hughes and E. Kosik, Virology 136:89-99, 1984; J. Sorge and S. H. Hughes, J. Mol. Appl. Genet. 1:547-599, 1982; J. Sorge, B. Ricci, and S. Hughes, J. Virol. 48:667-675, 1983), all of which derive from Rous sarcoma virus. In this report we describe three improvements in the earlier constructions. First, the vectors have been restructured as proviruses, which considerably improves the efficiency of virus production following acute transfection. Second, a series of miniplasmids have been developed, which we call adaptors, and these miniplasmids can be used to convert virtually any DNA segment into a ClaI fragment suitable for insertion into the retroviral (or other) vectors. Adaptors have been developed that supply regions of functional significance, including a splice acceptor and an initiator ATG. Finally, the region of env defining subgroup specificity, A in the original vectors, has been substituted by the corresponding regions of subgroup B and D viruses, giving vectors with additional subgroup specificities and increased host ranges.

Animals↗
PubMed1985

Structure and biological activity of human homologs of the raf/mil oncogene.

Two human genes homologous to the raf/mil oncogene have been cloned and sequenced. One, c-raf-2, is a processed pseudogene; the other, c-raf-1, contains nine exons homologous to both raf and mil and two additional exons homologous to mil. A 3' portion of c-raf-1 containing six of the seven amino acid differences relative to murine v-raf can substitute for the 3' portion of v-raf in a transformation assay. Sequence homologies between c-raf-1 and Moloney leukemia virus at both ends of v-raf indicate that the viral gene was acquired by homologous recombination. Although the data are consistent with the traditional model of retroviral transduction, they also raise the possibility that the transduction occurred in a double crossover event between proviral DNA and the murine gene.

Amino Acid Sequence↗
PubMed1984

3'-Terminal region of avian carcinoma virus MH2 shares sequence elements with avian sarcoma viruses Y73 and SR-A.

We determined the nucleotide sequence of the acute transforming avian retrovirus MH2 from an HgiAI site within the coding region of its oncogene, v-myc, to the KpnI site within the long terminal repeat. Comparison with published sequences from other retroviruses allowed us to identify all sequence elements in this region. We conclude that MH2 contains a unique assembly of 3'-terminal sequences, which includes part of the helper virus-derived SPC region of avian sarcoma virus Y73 and the complete F3 and F1 segments of Rous sarcoma virus strain SR-A.

Animals↗
PubMed1984

Oxygen toxicity in Treponema pallidum: deoxyribonucleic acid single-stranded breakage induced by low doses of hydrogen peroxide.

The effect of hydrogen peroxide on Treponema pallidum was investigated. The in vitro loss of virulence (as measured by rabbit inoculation) of T. pallidum was accelerated by as low as 100 microM hydrogen peroxide in the complex maintenance medium used. Higher doses led to rapidly accelerated death with 500 microM hydrogen peroxide causing sterilization of the medium within 3 to 4 h. Since hydrogen peroxide is known to cause single-stranded breaks in DNA, the effect of hydrogen peroxide on the treponemal genome was examined. Extensive breakage was caused by 100 microM hydrogen peroxide as determined on alkaline sucrose gradients. A limit was reached at 250 microM and above. Single-stranded breaks could be demonstrated as early as 5-10 min after exposure to hydrogen peroxide when the treponemes were exposed to 250 microM hydrogen peroxide; accelerated death was evident by 2 h past exposure demonstrating that DNA breakage was preceding death. Treponemal death caused by penicillin did not result in DNA breakage. The repair-proficient bacterium Escherichia coli K-12 was compared with T. pallidum. It required 10-100 times more hydrogen peroxide to cause various levels of breakage. Escherichia coli K-12 rapidly repaired DNA breakage once hydrogen peroxide was removed by addition of catalase. Treponema pallidum, in comparison, showed little or no repair in vitro. Addition of catalase or dithiothreitol to the medium protected against all but a low level of breakage; this may reflect on the ability of catalase and reducing agents to protect T. pallidum against oxygen toxicity in vitro.

DNA Repair↗