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Biomedical subjects

J Bag

Publications and source records attributed to J Bag.

At least 37 records · Page 2Linked to original sources
PubMed1988

Regulation of translation and stability of an mRNA coding for a 40-kDa polypeptide in rat L6 muscle cells.

The mRNA coding for a 40-kDa polypeptide (P-40) was previously cloned and sub-cellular distribution of this mRNA was examined in rat L6 myoblast cells under different conditions [Pramanik, S. & Bag, J. (1987) Eur. J. Biochem. 170, 59-67]. The translation of this mRNA was found to be regulated during differentiation of myoblasts. This mRNA was translated in proliferating myoblasts but not in the non-dividing differentiated myotubes. We have further examined whether the mRNA present in the polysomal fraction of myoblasts and that in the free non-polysomal fraction in myotubes was identical by nuclease S1 mapping. The coding strand of the 600-base-pair PstI fragment of the recombinant clone was 3'-end-labeled with cordycepin 5'-[alpha-32P]triphosphate and hybridized with RNA from either myoblasts or myotubes. The results of these studies have shown that RNA from both preparations was fully able to hybridize with the probe DNA and, therefore, protected the 600-nucleotide-long fragment from nuclease S1 digestion, thus suggesting that the sequence of 600 nucleotides at 3' ends of both translationally active polysomal mRNA of myoblasts and repressed free mRNA of myotubes are identical. These results also confirmed the results of our earlier studies on the subcellular distribution of this mRNA by Northern blot analysis. Further studies were also performed to determine whether withdrawal of muscle cells from the cell cycle during differentiation to form myotubes alone was responsible for regulating translation of P-40 mRNA. The results of the subcellular distribution of this mRNA in proliferating myoblasts following inhibition of DNA synthesis by cytosine arabinoside have shown that translation of P-40 mRNA continued in absence of DNA synthesis. This observation suggests that an additional signal is necessary to block the translation of P-40 mRNA in myotubes. The relationship between the translation of P-40 mRNA and its stability was examined. Two different methods were used to determine the stability of mRNAs. The first approach was by determining the steady-state levels of this mRNA following inhibition of RNA synthesis by actinomycin D. In the second method, we have determined the amount of 3H-labeled P-40 mRNA during pulse and chase experiments. Both methods produced similar results. It was found that the stability of P-40 mRNA was not altered during differentiation of rat L6 cells. The results of pulse and chase studies have also shown that P-40 mRNA was synthesized in both myoblasts and myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amanitins↗
PubMed1988

Developmentally regulated slow troponin C messenger RNA in chicken skeletal and cardiac muscles.

Regulation of slow troponin C gene expression was examined in both skeletal and cardiac muscle at various stages of development in chicken. The steady-state levels of troponin C mRNA were initially measured by Northern blot analysis. It was observed that the level of troponin C mRNA reached its maximum in both skeletal and cardiac muscle of 16- to 18-day-old embryos. A drop in troponin C mRNA level was observed just prior to hatching. The level of actin mRNA, myosin heavy chain mRNA, and mRNA for a nonmuscle protein, vimentin, was also similarly regulated during development of chicken muscles. Further studies were carried out to determine the level of slow troponin C mRNA using nuclease S1 protection analysis. A significant amount of slow troponin C mRNA was found in the skeletal muscle of adult chicken, which predominantly consists of the fast isoform of troponin C. This observation suggests the possibility of post-transcriptional control of slow troponin C synthesis in skeletal muscle. Primary cultures of cardiac myocytes were also used to determine how the troponin C mRNA level is regulated in a culture of cardiac muscle cells. Measurements of the steady-state levels of slow troponin C mRNA by nuclease S1 protection analysis show that it was maximal in 60-h-old cultures. A drop in the level of this mRNA was observed after these cells were maintained in culture for 4 days.

Actins↗
PubMed1987

Translation of an mRNA in rat L6 muscle cells is regulated within the cell cycle.

In muscle cells two populations of mRNA are present in the cytoplasm. The majority of mRNA is associated with ribosomes and active in protein synthesis. A small population of cytoplasmic mRNA occur as free mRNA-protein complex and is not associated with ribosomes. This apparently repressed population of mRNA from rat L6 myoblast cells was used to construct a cDNA library. Radioactively labeled cDNA preparations of polysomal and free (or repressed) mRNA populations showed that at least ten recombinant clones preferentially annealed to the cDNA from repressed mRNA. One of these clones was extensively studied. The DNA from a recombinant plasmid D12 hybridized to a 1.3-kb poly(A)-rich mRNA. In proliferating myoblast cells, the 1.3-kb mRNA was more abundant in the polysomal fraction and mostly free in the non-dividing myotubes. In contrast to this mRNA, 90% of alpha and beta actin mRNAs were translated in both myoblasts and myotubes. Further analysis of distribution of the 1.3-kb RNA in the polysomal (active) and free (repressed) fractions in fusion-arrested postmitotic myotubes suggested that fusion of myoblasts was not necessary for the control of translation of this mRNA. Withdrawal of muscle cells from the cell cycle appeared to be involved in regulating translation of this mRNA. The presence of this mRNA was not, however, limited to muscle cells. This mRNA was also present in the repressed state in rat liver and kidney cells. These results, therefore, suggest that the 1.3-kb mRNA is probably translated during a particular phase of the cell cycle and is not translated in terminally differentiated non-dividing cells. Messenger RNA homologous to the 600-base-pair insert of the recombinant plasmid D12 was isolated by hybrid selection procedure from both polysomal mRNA of myoblasts and free mRNA of myotubes. Translation of the hybrid selected mRNAs from both myoblasts and myotubes in rabbit reticulocyte lysate cell-free system synthesized a 40-kDa polypeptide. These results suggest that the repressed population of 1.3-kb mRNA can be translated in vitro. The hybridization pattern of DNA from the recombinant plasmid D12 with rat genomic DNA suggested that the 1.3-kb mRNA is derived from moderately repetitive rat DNA with a repetition frequency of approximately 100 copies per haploid genome.

Actins↗
PubMed1987

Regulation of troponin C synthesis in primary culture of chicken cardiac muscle cells.

Cardiac myocyte cell culture from fourteen day old embryonic chicken heart was prepared. This cultured cell system was used to examine the regulation of troponin C (TnC) synthesis in cardiac muscle. To examine the regulation of TnC polypeptide synthesis, cardiac myocyte cells were pulse labelled with 35S-methionine at different days after plating. The synthesis of TnC was measured by determining the amount of radioactivity incorporated into the TnC polypeptide following separation by two dimensional gel electrophoresis. These measurements showed that TnC synthesis was maximum in 36 to 48 h old cultures and reached its lowest level in 4 day old cultures. This was in contrast to the synthesis of actin and tropomyosin. Synthesis of these polypeptides were lowest in 36 to 48 h old cultures and was maximum in 7 day old cultures. To examine whether the synthesis of TnC polypeptide paralleled the levels of TnC mRNA the sequences homologous to quail slow TnC cDNA clone were measured by hybridisation. The results showed that the decrease in the synthesis of troponin C polypeptide cannot be fully explained by the decrease in the steady state level of troponin C mRNA. The possibility of a role of translational control of troponin C mRNA in this process is discussed.

Actins↗
PubMed1987

Attachment of mRNA to the cytoskeletal framework and translational control of gene expression in rat L6 muscle cells.

The mRNA of rat L6 muscle cells was distributed between a detergent-insoluble fraction containing the cytoskeletal framework and a detergent-soluble fraction. The majority of cytoskeleton-bound mRNA was translationally active and present as polysomes. The mRNA of the detergent-soluble fraction was not associated with the ribosomes and, thus, considered to be the repressed free population. The binding of mRNA was not mediated through ribosomes or the poly(A) region of mRNA. Cross-linking of RNA and proteins was used to examine whether proteins of the cytoskeletal framework were involved in binding mRNA to this structure. Analysis of the mRNA-protein complexes has shown that a large number of polypeptides of molecular masses between 15 and 220 kilodaltons (kDa) were associated with both cytoskeleton-bound and soluble mRNAs. However, a 165-kDa polypeptide was preferentially associated with cytoskeleton-bound mRNA-protein complexes. This polypeptide was also enriched in the total proteins of the cytoskeleton fraction. We have suggested a receptor-like role for the 165-kDa polypeptide in binding mRNA to the cytoskeletal framework. The mechanism of interaction between the cytoskeleton and mRNA was further examined by using a ghost-monolayer transcription system. The mRNA synthesized by this transcription system was preferentially retained in the detergent-insoluble cytoskeleton component of the ghost-monolayer preparation. To understand the physiological significance of the distribution of mRNA between the translationally active cytoskeleton-bound and repressed soluble fractions we have isolated a cDNA clone for a 1.3-kilobase (kb) mRNA. This mRNA was preferentially repressed in myotubes. Distribution of this mRNA was determined by Northern blot analysis using the recombinant plasmid. This analysis indicates that nearly 90% of this mRNA was not associated with ribosomes. In contrast, only 3% of alpha-actin mRNA was found in the repressed population. However, approximately 25% of the 1.3-kb mRNA was present as repressed free messenger ribonucleoprotein. This behaviour is again different from that of actin. All of the cytoskeleton-bound alpha-actin mRNA was associated with polysomes. Furthermore, most of the small amount of alpha-actin mRNA which was present in the soluble fraction was also associated with polysomes. We have, therefore, concluded from these observations that binding of mRNA to the cytoskeleton framework and translation of mRNA are two separate events. We have suggested that mRNA is transported to the cytoplasm as a cytoskeleton-associated complex and further interaction with ribosomes may stabilize this complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Association of messenger RNA with the cytoskeletal framework in rat L6 myogenic cells.

The distribution of mRNA between the detergent-soluble and insoluble (cytoskeleton) fractions in rat L6 myoblast and myotube cells was examined. Approximately 85% of cytoplasmic mRNA in both myoblasts and myotubes was found associated with the cytoskeletal framework. The cytoskeleton-bound mRNA was present as polysomes. In contrast, the mRNA of the detergent-soluble fraction was not associated with ribosomes and was thus considered to be the repressed population. The association of mRNA with the cytoskeletal framework was not affected by treatments leading to dissociation of polysomes. Differential distribution of mRNA between the soluble and cytoskeleton-bound fractions was analyzed by in vitro translation. The mRNAs coding for polypeptides of molecular masses 40 kDa and 60 kDa were preferentially enriched in the soluble fraction. The nature of binding between mRNA and the cytoskeletal framework was examined following in vivo cross-linking of RNA and protein by irradiating muscle cells with ultraviolet light. It was observed that this treatment covalently linked RNA and the neighbouring protein moieties without any detectable damage to the cytoskeletal framework, as measured by the distribution of RNAs and proteins between the cytoskeleton and soluble fractions. Analysis of the polypeptide moieties cross-linked to the mRNA have shown that a large number of polypeptides of molecular masses between 15-220 kDa were associated with both cytoskeleton-bound and soluble mRNAs. The polypeptide moieties of these mRNA-protein complexes were not only similar in the cytoskeleton and soluble mRNA-protein complexes but also were similar between myoblasts and myotubes. However, one polypeptide of 165 kDa was preferentially associated with the cytoskeleton-bound mRNA-protein complexes. Interestingly this 165-kDa polypeptide was also preferentially enriched in the total proteins from the cytoskeleton fraction. This result suggests a possible role of the 165-kDa polypeptide in association between mRNA and the cytoskeletal framework. To examine the mechanism of interaction between mRNA and the cytoskeletal framework we have reported here a ghost monolayer transcription system from myotubes. This transcription system was able to synthesize rRNA and mRNA. The mRNA transcribed in vitro was preferentially associated with the cytoskeleton structure present in the ghost monolayer system.

Binding Sites↗
PubMed1985

Recovery of normal protein synthesis in heat-shocked chicken myotubes by liposome-mediated transfer of mRNAs.

Exposure of chicken myotube culture to 45 degrees C induced the synthesis of three heat-shock polypeptides of 25 000, 65 000, and 81 000 daltons. Recovery to the normal pattern of protein synthesis was judged by the decrease in the synthesis of heat-shock polypeptides. This recovery to normal protein synthesis required de novo synthesis of mRNAs for normal cellular proteins. Inhibition of RNA synthesis by actinomycin D during recovery at 37 degrees C blocked the recovery process and resulted in the continued synthesis of heat-shock polypeptides. Large unilamellar vesicles were used to examine the effect of delivery of mRNAs isolated from both normal and heat-shocked myotubes on the recovery of these cells from heat-shock treatment. The results presented here show that liposome-mediated delivery of normal mRNAs to heat-shocked cells relieved the block of recovery by actinomycin. On the other hand, when mRNAs from heat-shocked cells were used during recovery, the synthesis of heat-shock polypeptides was stimulated. These observations suggest that the relative abundance of mRNAs in the cytoplasm plays a critical role in regulating protein synthesis in chicken myotube cultures.

Animals↗
PubMed1984

Cross-linked proteins associated with a specific mRNA in the cytoplasm of HeLa cells.

Cytoplasmic messenger RNAs of eukaryotic cells are distributed between polysomal and post-polysomal fractions (free) as protein-bound complexes. These studies were designed to determine whether a specific mRNA isolated from different subcellular compartments is complexed with the same family of polypeptides. As a first approach we have examined the proteins associated with mRNA which codes for histone H4. To perform these experiments HeLa cells were exposed to ultraviolet light to cross-link in vivo polypeptides which are closely associated with nucleic acid. To identify the polypeptides associated with mRNA specific for histones a genomic probe for histone H4 mRNA was immobilized on epoxy-cellulose. By hybrid selection specific mRNPs containing histone mRNA were isolated. Our results reveal the existence of a number of polypeptides associated with both polysomal and post-polysomal histone mRNAs. In polysomal histone mRNA two polypeptides of Mr = 49 000 and 52 500 were the major components. In contrast polypeptides of Mr = 43 000 and 57 000 were the major polypeptide components of post-polysomal (or free) histone mRNA. Furthermore, these results also suggest that the polypeptides associated with either polysomal or free H4 histone mRNA represent a subset of proteins found in poly(A)-free fractions or poly(A)-rich mRNA fractions.

Centrifugation, Density Gradient↗
PubMed1984

Cytoplasmic mRNA-protein complexes of chicken muscle cells and their role in protein synthesis.

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28 000, 32 000, 46 000, 65 000 and 150 000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130 000 and 43 000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81 000, 65 000 and 25 000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80 000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80 000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Regulation of heat-shock protein synthesis in chicken muscle culture during recovery from heat shock.

Exposure of chick myotube cultures to a temperature (45 degrees C) higher than their normal growing temperature (37 degrees C) caused extensive synthesis of three major polypeptides of Mr = 25 000, 65 000 and 81 000 referred to as 'heat-shock polypeptides' (hsps). When these cells were allowed to recover from heat-shock treatment at 37 degrees C for 6-8 h, the rate of accumulation of isotope into the 65 000-Mr and 81 000-Mr hsps declined to levels comparable to those in control cultures maintained at 37 degrees C. However, incorporation of isotope in the 25 000-Mr hsp continued at an elevated rate for a longer period than the 65 000-Mr and 81 000-Mr hsps. When heat-shocked cells were allowed to recover at 37 degrees C in the presence of actinomycin D to block new mRNA synthesis, the hsp synthesis as measured by the incorporation of radioactive isotope in these polypeptides continued at levels comparable to those in heat-shocked cells prior to recovery. The block of recovery by actinomycin D was due to the presence of a greater amount of functional hsp mRNAs in the polysomes as compared to untreated controls. The role of competition between the mRNAs for hsps and normal cellular proteins for the translation machinery in regulating protein synthesis during the recovery from heat shock has been discussed.

Animals↗
PubMed1983

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment.

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.

Animals↗
PubMed1982

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle.

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.

Animals↗
PubMed1981

Cytoplasmic nonpolysomal ribonucleoprotein complexes and translational control.

In this article, we discuss our attempts to establish the existence in the cytoplasm of regulatory molecules involved in translational control. Our studies have revealed the presence of cAMP independent protein kinase in the free mRNP complex capable of phosphorylating a Mr = 38 000 polypeptide, also part of the same complex. Both the kinase and the acceptor protein were found also as free proteins in the cytoplasmic pool. This kinase has been shown to be distinct from the heme regulated enzyme that phosphorylates the small subunit of eIF-2. Other regulatory molecules include small molecular weight RNAs found as part of an RNP complex. A 4S fraction isolated from this complex inhibited the translation of both capped and uncapped mRNAs in a cell-free protein synthesizing system. The biological role of the protein kinase and the 4S RNA fraction is considered.

Animals↗
PubMed1980

A cytoplasmic ribonucleoprotein complex containing a small RNA inhibitor of protein synthesis.

Ribonucleoprotein complexes (RNP) sedimenting between 10 and 15 S were isolated from the postpolysomal cytoplasmic fraction of embryonic chicken muscle. These RNP complexes lack mRNA but contain RNA with a sedimentation coefficient of 4.4 S. The 4.4 S RNA did not arise as a product of degradation during the course of the isolation procedure nor did it contain oligo(U)- or poly(A)-rich regions. Furthermore, the 4.4 S RNA-containing RNP complex was easily separable from free mRNPs and, therefore, is not considered as part of the free mRNP complexes. Both the 4.4 S RNA and 10 to 15 S RNP were able to inhibit translation of either "capped" or "uncapped" mRNA in a heterologous cell-free system. This inhibitory effect may result from interference of 4.4 S RNA with an early event in mRNA translation. A large number of polypeptides of Mr = 14,000 to 220,000 were present in the 10 to 15 S RNP. Among these, the most prominent polypeptides were of Mr = 36,000; 48,000; 52,000; 58,000; 65,000; 78,000; 84,000; 96,000; 105,000; 165,000; and 220,000. With the exception of the Mr = 36,000 polypeptide, these major components were also found in the nonpolysomal cytoplasmic mRNA protein complexes (free mRNP).

Animals↗
PubMed1979

The presence of protein kinase activity and acceptors of phosphate groups in nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle.

Nonpolysomal cytoplasmic (free) mRNA.protein (mRNP) complexes of embryonic chicken muscle were purified by a combination of oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The protein moieties of the purified mRNP complex were analyzed by two-dimensional gel electrophoresis using separation according to charge in the first dimension and molecular weight in the second. Sixteen polypeptides of Mr = 27,000 to 75,000 were present in the mRNP complex. These mRNP polypeptides displayed different electrophoretic migration properties than those of ribosomal proteins. A protein kinase activity was found associated with the mRNP. This enenzyme was able to transfer phosphate group(s) from ATP to at least three acidic mRNP polypeptides of Mr = 27,000, 38,000, and 73,000 and one basic polypeptide of Mr = 75,000. Among these, the Mr = 38,000 acidic polypeptide was the best acceptor of phosphate groups.

Animals↗