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D P Leader

Publications and source records attributed to D P Leader.

At least 55 records · Page 3Linked to original sources
PubMed1985

The same myosin alkali light chain gene is expressed in adult cardiac atria and in fetal skeletal muscle.

We have isolated from a cDNA library constructed using mouse cardiac mRNA sequences, a clone (pC6) homologous to part of the mRNA encoding the myosin alkali light chain MLC1A from adult mouse atria. This sequence also hybridizes to mRNA encoding the fetal light chain form MLC1emb expressed in both fused myotubes in culture and in 18 day fetal skeletal muscle. These mRNA sequences are indistinguishable from the MLC1A messenger both on the basis of size and of their thermal stability of hybridization. In vitro translation of mRNA selected by hybridization with pC6 results in a protein that comigrates with the fetal MLC1emb isoform, and two-dimensional gel electrophoresis of adult atrial and fetal skeletal muscle proteins shows MLC1A and MLC1emb to be indistinguishable in the mouse. Southern blot hybridization of clone pC6 to mouse genomic DNA and the analysis of restriction fragment length polymorphisms between different mouse species demonstrates the presence of a single hybridizing locus in the mouse genome. These data provide strong evidence that the atrial MLC1A and fetal skeletal MLC1emb isoform are encoded by the same gene and by the same mRNA and are thus identical proteins.

Animals↗
PubMed1985

The structure of a cloned mouse gamma-actin processed pseudogene.

The complete nucleotide (nt) sequence of a gamma-actin-like pseudogene (M gamma A-psi 1), isolated from a mouse genomic library in phage lambda, was determined. The pseudogene was shown to be of the processed type by the fact that it lacked introns, ended in a poly(dA) region, and was flanked by direct repeats. There were ten differences in the predicted amino acid (aa) sequence from that of the authentic nonmuscle gamma-actin. An unusual feature of M gamma A-psi 1 was the complete absence of DNA corresponding to the 5' end of the mRNA up to the nt preceding the Ala codon at aa position 7. This suggests that M gamma A-psi 1 originated from a truncated mRNA or from an incomplete reverse transcript.

Actins↗
PubMed1985

Genes for skeletal muscle myosin heavy chains are clustered and are not located on the same mouse chromosome as a cardiac myosin heavy chain gene.

Myosin heavy chain (MHC) genes are expressed as several distinct isoforms in a tissue- and stage-specific manner; three skeletal muscle MHC isoforms appear sequentially during development. We have isolated cDNA clones, identified by RNA blot hybridization and by nucleotide sequence determination as coding for portions of the embryonic (pMHC2.2), perinatal (pMHC16.2A), and alpha(V1) cardiac (pMHC141 and pMHC101) MHC isoforms. These four probes and the adult skeletal MHC probe (pMHC32) have been used on Southern blots of genomic DNA to detect restriction fragment length polymorphisms defining the alleles for these genes in mouse species Mus musculus and Mus spretus. In this way, we followed the segregation of skeletal and cardiac MHC genes in 42 offspring resulting from an interspecies backcross. We found that the embryonic, perinatal, and adult skeletal MHC genes are clustered on chromosome 11 near the locus nude, the skeletal and cardiac MHC genes do not cosegregate, and the alpha(V1) cardiac MHC gene is located on chromosome 14 close to Np-1. This result is in contrast to that for other contractile protein genes such as the alkali myosin light chain and the actin multigene families, which are dispersed in the genome.

Animals↗
PubMed1985

Protein kinase activities associated with the virions of pseudorabies and herpes simplex virus.

Protein kinase has been extracted in soluble form from virions of pseudorabies virus using 10% NP40, 0.6 M-NaCl. Chromatographic analysis of the extract on DEAE-cellulose and on phosphocellulose showed it to contain more than one kinase. The activity responsible for the phosphorylation of the major phosphoproteins (mol. wts. 120 000, 115 000 and 72 000) of virions was found to be similar in its properties to the host enzyme casein kinase II. Purified casein kinase II from ascites cells or from pig liver was able to phosphorylate heat-inactivated virions. In addition to the major phosphoproteins, active virion preparations were able to phosphorylate a minor low molecular weight phosphoprotein, incorporation into which could be stimulated by the addition of cyclic AMP to the assay. Purified host cyclic AMP-dependent protein kinase also phosphorylated this protein in heat-inactivated virions. Analysis of herpes simplex virus type 1 showed that the major phosphoproteins (VP12 and VP23) could be phosphorylated in heat-inactivated virions by added casein kinase II. One of these (VP12) together with a further minor phosphoprotein (VP14) could be phosphorylated by cyclic AMP-dependent protein kinase.

Animals↗
PubMed1984

Heat shock causes diverse changes in the phosphorylation of the ribosomal proteins of mammalian cells.

When HeLa cells or BHK cells were subjected to heat shock at 42 degrees C (for 2 h) or 45 degrees C (for 10 min) there was extensive dephosphorylation of ribosomal protein S6. Concomitantly ribosomal protein L14, which is not significantly phosphorylated in normal cells, became phosphorylated, as did a non-structural protein of Mr = 27000, associated with the ribosomes. The latter effects were not prevented by cycloheximide or actinomycin D. When cells shocked at 45 degrees C for 10 min were returned to 37 degrees C for 2 h there was rephosphorylation of ribosomal protein S6 and dephosphorylation of the 27 kDa protein, but not of ribosomal protein L14.

Animals↗
PubMed1984

The phosphorylation of ribosomal protein S6 in hamster fibroblasts infected with pseudorabies virus inactivated by ultraviolet radiation.

Infection of baby hamster fibroblasts with pseudorabies virus at high multiplicities resulted in a substantial increase in the phosphorylation of ribosomal protein S6. However, the phosphorylation was still observed with virus that had been completely inactivated by u.v. irradiation. We therefore conclude that expression of the viral genome is not required for the virus to elicit this effect.

Animals↗
PubMed1983

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells.

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.

Adenosine Triphosphate↗
PubMed1981

The protein synthetic activity in vitro of ribosomes differing in the extent of phosphorylation of their ribosomal proteins.

We describe a re-examination of the cell-free protein synthetic activity of eukaryotic ribosomes having proteins phosphorylated to different extents. Ribosomal 40 S subunits were isolated both from a variety of cells in which there is relatively little phosphorylation of ribosomal protein S6, and from cells subjected in vivo to different stimuli that promote the extensive phosphorylation of protein S6. The ability of these subunits to bind Met-tRNA as well as the second amino acyl-tRNA (Val-tRNA) was compared in the presence of highly purified initiation factors, elongation factor EF-1 at various concentrations of 60S subunits, 9 S globin mRNA and potassium ions. The ability of the subunits to synthesize polyphenylalanine was also studied using highly purified elongation factors. In no case was any significant difference in activity observed between ribosomes with protein S6 phosphorylated to different extents. Similar, though less extensive, studies were preformed comparing 60 S ribosomal subunits differing in the extent of phosphorylation of the acidic phosphoprotein, L gamma , and of L14. No difference in activity was observed between these ribosomes.

Animals↗
PubMed1981

Increased phosphorylation of a liver ribosomal protein in mice carrying ascites tumours.

The extent of phosphorylation of ribosomal protein S6 in the livers of mice carrying the Krebs II ascites tumour has been examined by two-dimensional gel electrophoresis. Seven days after injection, an increase was observed in the amount of stained material migrating at the position of the phosphorylated derivatives of ribosomal protein S6, and there was also an increase in the proportion of ribosomes sedimenting as monosomes. The extent of phosphorylation of ribosomal protein S6 in the ascites cells themselves was low. The findings are discussed in relation to the phosphorylation of ribosomal protein S6 in transformed cells and in other pathological states of the liver.

Animals↗
PubMed1981

Phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus.

In BHK cells infected with pseudorabies virus, there was a substantial increase in the phosphorylation of ribosomal protein S6. This increase occurred between 2 and 4 h after infection and persisted at least until 9 h. We estimated that in mock-infected cells S6 contained, on an average, one phosphate group per protein chain, whereas in infected cells this rose to between four and five phosphate groups per protein chain. A second ribosomal protein, either S16 or S18, was also phosphorylated after infection. No increase in cyclic AMP was found at the time of phosphorylation. We also found an increased phosphorylation of S6 in herpes simplex virus-infected BHK cells.

Adenosine Triphosphate↗
PubMed1980

The phosphorylation of protein S6 in the newly-synthesized cytoplasmic ribosomes of hamster fibroblasts.

Ribosomes were isolated from baby hamster kidney fibroblasts, either 20 min or 2 days after labelling with radioactive amino acids, and their proteins subjected to two-dimensional polyacrylamide gel electrophoresis. No significant differences were observed between the amounts of radioactivity associated with the position of the phosphorylated derivatives of protein S6. This suggests that the phosphorylation is unlikely to be important in ribosomal biogenesis or extranuclear transport.

Animals↗
PubMed1980

Phosphorylated and other modified forms of eukaryotic ribosomal protein S3 analysed by two-dimensional gel electrophoresis.

Proteins were isolated from the 40S ribosomal subunits of baby-hamster kidney fibroblasts and subjected to two-dimensional gel electrophoresis. When the cells were pretreated with cyclic AMP or 2-deoxyglucose a more basic derivative of ribosomal protein S3 or S3a was often observed, apparently similar to that previously reported to occur early in liver generation. This derivative was not a dephosphorylated form of protein S3, which protein does not appear to be phosphorylated in normal cells; nor did it correspond to the proteolytic fragment, S3b. It appears to be an oxidation product of protein S3 or S3a, as it can be eliminated by thorough reduction of the ribosomal protein before electrophoresis. In contrast with previous results with Krebs II ascites cells, starvation of baby-hamster kidney fibroblasts of glucose did not cause extensive phosphorylation of ribosomal protein S3.

Animals↗
PubMed1980

The effect of diabetes and insulin on the polyadenylic acid-containing RNA of rat skeletal muscle.

Poly(A)-containing RNA in various fractions of RNA from rat skeletal muscle has been detected and quantitated by hybridization to [3H]poly(U). Comparison has been made between the RNA in skeletal muscle from normal adult rats, from rats 2 days after induction of diabetes with streptozotocin, and from diabetic rats killed 60 min after injection of insulin. The poly(A)-containing RNA constituted a similar proportion of the total RNA in skeletal muscle from each of the three types of rats. In diabetes there was a decrease in the proportion of skeletal muscle ribosomes sedimenting as polyribosomes, but this was reversed after rats had been injected with insulin. However these changes were not associated with any alterations in the relative amounts of poly(A)-containing RNA in ribosomes isolated from rat skeletal muscle. Diabetes did not significantly alter the size distribution of the poly(A)-containing RNA or its poly(A) segment. If it is assumed that poly(A)-containing RNA is mRNA, these results imply that insulin stimulates protein synthesis in the skeletal muscle of diabetic rats by affecting the translation of pre-existing mRNA. Sucrose density gradient analysis showed that there was less poly(A)-containing RNA in the polyribosome region from diabetic rats than there was in that from normal rats. The balance of the poly(A)-containing RNA in the ribosomes from diabetic rats (presumed to be the untranslated mRNA) was found associated with rapidly sedimenting aggregated ribosomes, but its native form has not yet been determined.

Animals↗
PubMed1980

The anomalous migration during two-dimensional gel electrophoresis of eukaryotic ribosomal proteins with oxidised thiol groups.

Two-dimensional gel electrophoresis of eukaryotic ribosomal proteins under conditions in which thiol groups are not maintained in a reduced state has been found to cause marked changes in the migration of certain proteins, apparently due to the formation of intramolecular disulphide bridges. The results suggest that certain previously designated eukaryotic ribosomal proteins are not, in fact, unique species.

Animals↗
PubMed1978

The acidic ribosomal phosphoprotein of eukaryotes and its relationship to ribosomal proteins L7 and L12 of Escherichia coli.

The acidic ribosomal phosphoprotein, Lgamma, of Krebs II ascites cells was further characterized and compared with proteins L7 and L12 of Escherichia coli. Ribosomal protein Lgamma was selectively removed from 60S sibosomal subunits by 50% ethanol and 1M-NH4Cl, and antibodies raised against protein Lgamma cross-reacted with E. coli protein L12 in immunodiffusion experiments. These and other, previously reported, data support the proposal that the uekaryotic counterpart of E. coli proteins L7 and L12 is phosphorylated.

Cell Line↗
PubMed1978

Codon-specific interaction of uncharged transfer-RNA with eukaryotic ribosomes.

Rat liver ribosomes bound [32P]tRNAPhe in both a codon-dependent and codon-independent manner. The codon-dependent binding was studied further by utilising the ability of the unchanged tRNAPhe to inhibit the poly(U)-directed binding of [3H]Phe-tRNA to ribosomes. At least part of the codon-dependent binding of uncharged tRNA appears to be to the ribosomal A-site.

Binding Sites↗