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R Jove

Publications and source records attributed to R Jove.

89 records · Page 5Linked to original sources
PubMed1987

Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region.

Cellular src protein, p60c-src, is phosphorylated on tyrosine 527 in chicken embryo fibroblasts, and this phosphorylation is implicated in suppressing the protein-tyrosine kinase activity and transforming potential of p60c-src. To determine whether tyrosine 527 phosphorylation is dependent on p60c-src kinase activity, the ATP-binding site of chicken p60c-src was destroyed by substitution of lysine 295 with methionine. The resultant protein, p60c-src(M295), expressed either in chicken cells or in yeast, lacked detectable kinase activity. Nevertheless, tyrosine and serine phosphorylation of p60c-src(M295) overproduced in chicken cells were indistinguishable from that of authentic p60c-src. By contrast, p60c-src(M295) was not phosphorylated on tyrosine in yeast. These results suggest that a protein kinase present in chicken cells but not in yeast phosphorylates tyrosine 527 in trans, and are consistent with the possibility that this kinase is distinct from p60c-src.

Adenosine Triphosphate↗
PubMed1987

RNA polymerase II terminates transcription in vitro in the SV40 origin region.

To begin to study signals on DNA that can cause mammalian RNA polymerase II to terminate transcription, we examined the RNA transcripts produced from a number of different DNA templates in a HeLa whole-cell extract. When transcripts initiating from the strong adenovirus late promoter and extending through the SV40 promoter-replication origin region were analyzed, it was observed that a significant fraction (approximately 75%) were terminated within these SV40 sequences. These transcripts, the 3' ends of which were mapped to several sites within an approximately 200 base pair region, appeared to result from transcription termination, as judged by kinetic and pulse-chase experiments. The possible significance of these findings with respect to SV40 gene expression in particular and transcription termination in general are discussed.

Adenoviruses, Human↗
PubMed1987

Characterization of avian and viral p60src proteins expressed in yeast.

Avian and viral p60src proteins were expressed from a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. Both the viral and cellular src proteins produced in yeast cells were myristoylated at their amino termini, as is the case for src proteins expressed in chicken embryo fibroblasts. The viral src protein produced in yeast autophosphorylated at tyrosine-416 in vivo and had approximately the same level of in vitro kinase activity as p60v-src expressed in Rous sarcoma virus-transformed cells. Unlike p60c-src expressed in chicken cells, which is phosphorylated on tyrosine in vivo almost exclusively at tyrosine-527, p60c-src expressed in yeast was phosphorylated 2.5-3 times more at tyrosine-416 than at tyrosine-527. The specific activity of the p60c-src produced in yeast was 2.5-5.0 times higher than that of p60c-src overexpressed from a retroviral vector in chicken cells, implicating the altered state of in vivo phosphorylation in modulation of the in vitro kinase activity. The expression of p60v-src substantially slowed down the growth of the yeast cells, suggesting that phosphorylation of yeast proteins essential for cell growth may have interfered with their proper functioning.

Animals↗
PubMed1987

Analysis of p60v-src mutants carrying lesions involved in temperature sensitivity.

Analysis of the src genes of three temperature-sensitive (ts) mutants of Rous sarcoma virus (tsNY68, tsNY72-4, and PA104) showed that each has two C-terminal mutations in the kinase domain required for temperature sensitivity, as assayed by morphological alteration and anchorage-independent growth. In all three mutants, one of the mutations is a valine-to-methionine change at position 461. To assess the contribution of each mutation to the biochemical properties of the src protein, we analyzed the kinase activity and the interaction with cellular proteins p50 and p90 of recombinant src gene products in which only one mutation was combined with wild-type src sequences. Chimeric src protein containing only the Met-461 mutation was indistinguishable from the wild type by all criteria examined, while the effect of the second C-terminal mutation alone varied with the defectiveness of the parental ts mutant. The second mutation alone, while not sufficient to cause ts transformation, altered p60src complex formation with cellular proteins p50 and p90 and altered the in vitro thermolability of src kinase activity. The results indicate that these biochemical properties of p60src are more sensitive to mutation than others, such as in vivo kinase activity, which require more profound structural alterations.

Animals↗
PubMed1987

The membrane-binding domain and myristylation of p60v-src are not essential for stimulation of cell proliferation.

Previous studies showed that the amino-terminal domain of Rous sarcoma virus p60v-src involved in myristylation and membrane association of the protein is required for morphological transformation and anchorage independence. Analysis of src delection mutants revealed that the amino-terminal one-third of p60v-src, including the membrane-binding domain, is not essential for induction of cell proliferation. These results demonstrated that, in contrast to the cellular target(s) involved in morphological transformation and anchorage independence, the target(s) involved in mitogenic activity is accessible to nonmyristylated src proteins.

Animals↗
PubMed1986

Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters.

The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology.

Amino Acid Sequence↗
PubMed1986

Biochemical properties of p60v-src mutants that induce different cell transformation parameters.

PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation.

Animals↗
PubMed1986

Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants.

The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.

Amino Acid Sequence↗
PubMed1985

Lack of induction of neuroretinal cell proliferation by Rous sarcoma virus variants that carry the c-src gene.

Expression of p60v-src of Rous sarcoma virus in cultured chicken embryo neuroretinal cells was previously shown to result in the transformation and sustained proliferation of normally quiescent cell populations. We show here that Rous sarcoma virus variants that encode p60c-src, the cellular homolog of p60v-src, lack the ability to induce morphological transformation and cell proliferation of cultured neuroretinal cells. Neuroretinal cells infected with c-src-containing viruses, however, possess no less p60 protein kinase activity assayed in the immune complex than those infected with the transformation-defective Rous sarcoma virus mutants PA101 or PA104, which do stimulate the growth of these cells.

Animals↗
PubMed1984

In vitro transcription from the adenovirus 2 major late promoter utilizing templates truncated at promoter-proximal sites.

We report that adenovirus 2 DNA sequences located between positions-66 and -51 upstream of the major late cap site (position +1) enhance transcription initiation from this promoter by up to 5- to 10-fold in HeLa whole cell lysates. This enhancing effect is template concentration-dependent and is abolished by truncation of the template immediately upstream of -66. Additionally, specific transcripts are not detected from templates truncated at +33 downstream of the cap site. These results define a minimum region of approximately 100 base pairs encompassing the transcription start site that appears to interact with the RNA polymerase II transcription complex during initiation. Analysis of the shortest runoff transcripts that can be synthesized in vitro revealed that RNAs as short as 50 nucleotides are quantitatively modified by guanylylation and methylation to cap 1 structures. In contrast, short RNAs containing guanylylated but unmethylated cap structures are not efficiently utilized as substrates by endogenous cap-methylating enzymes in the HeLa lysate. These findings, together with the observation that the synthesis of short transcripts is sensitive to the presence of the methyltransferase inhibitor S-adenosylhomocysteine, suggest that cap formation is a promoter-proximal event that occurs concomitantly with the synthesis of a nascent RNA polymerase II transcript.

Adenoviruses, Human↗
PubMed1984

Transcription of methylated eukaryotic viral genes in a soluble in vitro system.

SV40 and adenovirus-2 (Ad2) recombinant plasmids containing long segments of poly(dC-dG) cloned adjacent to transcription control regions were methylated in vitro with Hbal methylase and transcribed in a soluble in vitro system. The addition of up to 40 or more base pairs of poly(m5dC-dG) immediately upstream or downstream of promoter regions was shown to have no effect on the accuracy or efficiency of specific transcription from these promoters in vitro. Methylation at various naturally occurring C-G sequences within or near these promoters also had no effect on transcription in vitro. The significance of these results with respect to possible mechanisms whereby DNA methylation might regulate eukaryotic gene expression is discussed.

Adenoviruses, Human↗
PubMed1984

Splicing pathways of SV40 mRNAs in X. laevis oocytes differ in their requirements for snRNPs.

To examine the role of small nuclear ribonucleoproteins (snRNPs) in mRNA splicing, we have injected SV40 DNA, in the presence or absence of anti-Sm or anti-(U1)RNP antibodies, into the nucleus of X. laevis oocytes, and analyzed the viral specific RNAs and proteins that were synthesized. In the absence of antibodies, the majority of the viral mRNAs were spliced, giving rise to transcripts and proteins analogous to those found in infected monkey cells. However, the relative efficiencies with which the various splice sites were utilized were different in the two cell types. When sera from systemic lupus erythematosus (SLE) patients containing anti-Sm or anti-(U1)RNP antibodies were coinjected with the viral DNA, splicing of L-strand-specific (late) mRNA was dramatically inhibited. Cleavage at both 5' and 3' splice sites was blocked, leading to an accumulation of unspliced primary transcripts. Neither the total amount of late RNA synthesized nor the formation of mature polyadenylated late mRNA 3' ends was affected. These results indicate that U1 snRNPs play a crucial role in mRNA splicing in vivo. Unexpectedly, the effects of the sera on E-strand-specific (early) viral mRNA splicing were different. All anti-Sm or -(U1)RNP sera tested had no detectable effect on the splicing of the mRNA coding for the small tumor antigen. A subset of these sera, however, inhibited large tumor antigen mRNA splicing. On the basis of these data it is suggested that different pre-mRNAs, or even different splice sites within the same pre-mRNA, have dissimilar interactions with snRNP particles in the splicing reaction.

Animals↗
PubMed1982

Transcription initiation by RNA polymerase II is inhibited by S-adenosylhomocysteine.

Most eukaryotic mRNAs are blocked at their 5' termini by guanylylation and methylation. These "cap structures" have been shown to play important roles in increasing the stability and translatability of mRNAs. Previous in vitro and in vivo data suggest that these modifications occur extremely early in the synthesis of RNA transcripts by RNA polymerase II. Here we show that S-adenosylhomocysteine (AdoHcy), both a product and an inhibitor of transmethylation reactions, inhibits transcription initiation by RNA polymerase II, but not by RNA polymerase III, in a HeLa whole-cell lysate. AdoHcy must be present during initiation to inhibit transcription and does not affect elongation by RNA polymerase II or the stability of the resultant transcript. Furthermore, AdoHcy does not inhibit transcription by purified HeLa RNA polymerase II. These results suggest that formation of the 5'-cap structure is coupled to initiation of transcription and is consistent with a close association between the capping enzymes and RNA polymerase II at the time of initiation.

Adenoviruses, Human↗
PubMed1980

Direct radioimmunoassay of nuclear 3,5,3' triiodothyronine in rat anterior pituitary.

Previous tracer studies have suggested that 5'-monodeiodination of l-thyroxine (T(4)) in anterior pituitary may contribute a substantial portion of specifically bound nuclear 3,5,3' l-triiodothyronine (T(3)) in this tissue in rats. To evaluate this possibility, a radioimmunoassay for nuclear T(3) in individual anterior pituitaries was developed. Animals received [(125)I]T(3) 60 min before removal of the anterior pituitary and isolation of the nuclei by differential centrifugation. This allowed calculation of the nuclear:serum T(3) ratio and comparison of expected with measured T(3). T(3) was extracted in ethanol, dried, and reconstituted in assay buffer. In untreated hypothyroid rats, anterior pituitary nuclear T(3) was 0.18 +/- 0.06 pg/mug DNA which was 0.13 pg/mug DNA greater than expected from the serum T(3) concentration and the pituitary nuclear:serum [(125)I]T(3) ratio. In 10 hypothyroid rats given a single bolus of 400 ng T(3)/100 g body wt., the nuclear T(3) by radioimmunoassay was 1.0 +/- 0.06 pg/mug DNA, whereas that expected from the T(3) specific activity calculations was 0.85 pg/mug DNA (P < 0.025). Serum T(4) concentrations in these rats were < 0.25 mug/dl but the nuclear T(3) derived from as little as 0.2 mug/dl T(4) could explain a large portion of these small discrepancies between observed and measured nuclear T(3). In 29 normal rats, anterior pituitary nuclear T(3) was 0.63+/-0.04 pg/mug DNA, whereas that expected from the serum T(3) concentration (55+/-2 ng/dl) was 0.23+/-0.02 pg/mug DNA (P < 0.001). Total pituitary T(3) based on this measurement was 92+/-6 pg. Because the maximal nuclear binding capacity for T(3) in rat anterior pituitary is 0.77 pg/mug DNA, these results suggest there is 82% occupancy of these nuclear receptors. The requirement for normal serum concentrations of both T(4) and T(3) to achieve normal nuclear T(3) saturation in anterior pituitary is in marked contrast to the situation in liver, kidney, and heart muscle which appear to require only a normal serum T(3). As a consequence, the anterior pituitary can monitor both serum T(4) and T(3) and respond appropriately to changes in their concentrations.

Animals↗