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M Y Fiszman

Publications and source records attributed to M Y Fiszman.

At least 37 records · Page 2Linked to original sources
PubMed1992

In vivo splicing of the beta tropomyosin pre-mRNA: a role for branch point and donor site competition.

The chicken beta tropomyosin gene contains two sets of alternatively spliced, mutually exclusive exons whose utilization is developmentally regulated. Exons 6A and 6B are used in nonmuscle cells (or undifferentiated muscle cells) and skeletal muscle cells, respectively. A complex arrangement of cis-acting sequence elements is involved in alternative splicing regulation. We have performed an extensive mutational analysis on the sequence spanning the region from exon 6A to the constitutive exon 7. A large number of mutant minigenes have been tested in transfection assays of cultured myogenic cells, and the splicing products have been analyzed by cDNA polymerase chain reaction. We demonstrate that in undifferentiated myoblasts, exon 6B is skipped as a result of a negative control on its selection, while exon 6A is spliced as a default choice. We provide evidence that the focal point of such a regulation is localized in the intron upstream of exon 6B and probably involves the blockage of its associated branch point. In differentiated myotubes, in contrast, both exons are accessible to the splicing machinery. We show that the preferential choice of exon 6B in this splicing environment depends on the existence of a competition between the two exons for the flanking constitutive splice sites. We demonstrate that both the donors and the branch points of the two exons are involved in this competition.

Animals↗
PubMed1992

Cis regulating elements which control in vivo alternative splicing of the chicken beta tropomyosin primary transcript.

The beta tropomyosin gene of the chicken contains a pair of alternatively spliced mutually exclusive exons the use of which is developmentally regulated. Exon 6A is used by non muscle and undifferentiated muscle cells (myoblasts) while exon 6B is exclusively used in differentiated skeletal muscle cells. A complex array of cis acting sequence elements are involved in the regulation of this alternative splicing process. Transfection assays of quail muscle cells in culture were used to define these cis acting elements. We show that, in undifferentiated muscle cells, exon 6B is skipped as a result of a negative control on its selection while exon 6A is spliced as a default choice. We provide evidence that this negative control involves a secondary structure of the primary transcript around the 5' end of exon 6B as well as intronic sequence elements located between the branch point and the acceptor splice site of exon 6B. In differentiated muscles, both exons are accessible to the splicing machinery and the preferential use of exon 6B depends on the existence of a competition between the two exons for the selection of the flanking splice sites. In particular, we show that the donor splice site of exon 6A is a weak splice site while the branch point associated with exon 6B is a strong branch point.

Alternative Splicing↗
PubMed1991

Characterization of muscle and non muscle Xenopus laevis tropomyosin mRNAs transcribed from the same gene. Developmental and tissue-specific expression.

We have isolated and characterized three Xenopus laevis tropomyosin cDNAs (XTM alpha 2, XTM alpha 4 and XTM05). XTM alpha 2 and XTM alpha 4 were isolated from a stage-42 embryo cDNA library and XTM05 from an oocyte cDNA library. XTM alpha 2 and XTM alpha 4 both code for a 284-amino-acid protein homologous to the skeletal alpha tropomyosin but diverge in their 3' untranslated sequences. This divergence is due to an absence of splicing in the early embryo. XTM05 codes for a 248-amino-acid non-muscle tropomyosin. Sequence analysis indicates that the three cDNAs are generated from a single gene which uses two promoters and possibly three sets of alternate exons. Northern blot analysis, with specific probes against each cDNA, was used to study the expression of the X. laevis alpha tropomyosin gene during development and in specific tissues. This analysis showed a different temporal usage of the two promoters and characterized the tissue-specific expression of the different transcripts.

Amino Acid Sequence↗
PubMed1991

The chicken gene encoding the alpha isoform of tropomyosin of fast-twitch muscle fibers: organization, expression and identification of the major proteins synthesized.

The chicken gene alpha fTM encoding the alpha-tropomyosin of fast-twitch muscle fibers (alpha fTM) covers 20 kb and consists of 15 exons. From this gene, three types of mature transcripts (1.3 kb, 2 kb and 2.8 kb) are expressed through the use of alternative promoters, alternatively spliced exons and multiple 3' end processing. Northern analysis and S1 mapping have shown that the 1.3-kb transcript (exons 1a, 2b, 3, 4, 5, 6b, 7, 8, 9a-9b) is expressed in fast-twitch skeletal muscles and that 2-kb transcripts are expressed in smooth muscle (exons 1a, 2a, 3, 4, 5, 6b, 7, 8, 9d) and in fibroblasts (exons 1a, 2b, 3, 4, 5, 6a or 6b, 7, 8, 9d). These 2-kb transcripts encode distinct proteins which we have identified by two-dimensional (2D) gel electrophoresis. The 2.8-kb transcript which has not been so far characterized in birds is expressed in brain (exons 1b, 3, 4, 5, 6b, 7, 8, 9c-9d). This transcript has been characterized by a cDNA polymerase chain reaction assay and by S1 nuclease mapping. It produces a major TM isoform of chick brain which we have identified by 2D gels.

Amino Acid Sequence↗
PubMed1991

Tissue-specific splicing in vivo of the beta-tropomyosin gene: dependence on an RNA secondary structure.

The beta-tropomyosin gene in chicken contains two mutually exclusive exons (exons 6A and 6B) which are used by the splicing apparatus in myogenic cells, respectively, before (myoblast stage) and after (myotube stage) differentiation. The myoblast splicing pattern is shown to depend on multiple sequence elements that are located in the upstream intron and in the exon 6B and that exert a negative control over exon 6B splicing. This regulation of splicing is due, at least in part, to a secondary structure of the primary transcript, which limits in vivo the accessibility of exon 6B in myoblasts.

Animals↗
PubMed1990

A nonmuscle tropomyosin is encoded by the smooth/skeletal beta-tropomyosin gene and its RNA is transcribed from an internal promoter.

The smooth/skeletal muscle beta-tropomyosin gene contains an additional exon (exon 1') which is located between exons 2 and 3 and which is used to generate a 1.3-kb transcript expressed in undifferentiated muscle as well as nonmuscle cells. This mRNA, besides exon 1', corresponds to exons 3, 4, 5, 6A, 7, 8, and 9B of the gene and codes for a 247-amino acid low molecular weight tropomyosin. Exon 1' contains the coding sequence for the first 44 amino acids of the protein as well as the whole 5'-untranslated region. During the transition from myoblasts to myotubes, initiation of transcription continues from the internal promoter but also occurs from the distal promoter in order to give rise to the skeletal beta-tropomyosin-specific transcript.

Amino Acid Sequence↗
PubMed1990

Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the beta tropomyosin gene.

The beta tropomyosin gene contains two internal exons which are spliced in a mutually exclusive manner. Exon 6B is specifically included in the mature transcripts expressed in skeletal muscle or cultured myotubes, while exon 6A is a myoblast- or smooth muscle-specific exon. The intron between them, which is never spliced in normal conditions, contains two characteristic features: first, the unusual location of the branch point at position -105 from the acceptor, and second, the presence of a very long pyrimidine stretch upstream of the skeletal muscle exon. In this study we designed a number of sequence modifications to investigate the role of these two elements and of a computer-predicted secondary structure in the mutually exclusive splicing of the two exons. We found that mutations in the skeletal exon as well as in the upstream intron could change in vivo the tissue-specific pattern as well as the mutually exclusive character of the two exons. Our results suggest that the unusual position of the branch point does not prevent the utilization of exon 6B in myoblasts and that the region around the acceptor site of exon 6B and the polypyrimidine tract have an important role in this control. Last, we discuss the possible implications of secondary structures.

Animals↗
PubMed1989

A subfragment of the beta tropomyosin gene is alternatively spliced when transfected into differentiating muscle cells.

A subgenomic fragment of the chicken beta tropomyosin gene which contains two alternative exons flanked by common exons was isolated and placed under the control of the SV 40 early promoter. This construction was subsequently used to transfect quail myoblasts together with a Neomycin resistance gene, and to isolate stable transfectants. mRNAs were isolated before and after differentiation and analyzed using a modification of the primer extension method. We show that myoblasts accumulate transcripts which contain the non muscle specific exon joined to the common exons while myotubes accumulate transcripts containing the muscle specific exon. These results, therefore demonstrate that such a subgenomic fragment contains all the necessary information to direct a correct developmentally regulated mutually exclusive splicing. They also strongly suggest that trans acting factors must be involved in the switch of the splicing pattern which takes place during the transition from myoblasts to myotubes. The same regulation cannot be faithfully reproduced during transient expression, since no difference in the use of exons 6A/6B is observed during differentiation and two aberrant minor splicing products are obtained which contain or lack both exons. We suggest that failure of exon 6A to splice to exon 6B is due to the existence of some structural constraints which lower the efficiency with which the intron between them is excised.

Animals↗
PubMed1989

A single gene codes for the beta subunits of smooth and skeletal muscle tropomyosin in the chicken.

A chicken genomic DNA library was screened with a full length cDNA corresponding to the beta subunit of smooth muscle tropomyosin. When hybridized with RNAs isolated from various tissues, this cDNA recognizes two mRNA species: one of 1.3 kilobase pairs present only in smooth muscle and one of 1.6 kilobase pairs present only in skeletal muscle. Two overlapping recombinant phages were shown to contain the entire locus and were further characterized. This locus contains 11 exons and spans approximately 13 kilobase pairs. Exon 1 (amino acids 1-38) contains the 5'-untranslated region which is common to the two mRNAs. Exons 6 (amino acids 189-213) and 11 (amino acids 258-284) contain sequences which are present exclusively in the 1.3-kilobase pair smooth muscle mRNA while exons 7 and 10, which code for an analogous region, contain sequences which are present exclusively in the 1.6-kilobase pair skeletal muscle mRNA (exons 10 and 11 also contain the entire 3'-untranslated regions of the corresponding mRNAs). Other exons, 2 to 5 (amino acids 39-188) and 8 and 9 (amino acids 214-257), contain sequences which are present in both mRNAs. Our results indicate that both the smooth and skeletal beta-tropomyosin mRNAs are derived from transcripts of a single gene with a unique promoter by a differential splicing mechanism.

Amino Acid Sequence↗
PubMed1989

Expression of alpha and beta tropomyosin subunits during early myogenesis in somites and limb buds of chick embryos.

The appearance of alpha and beta subunits of skeletal tropomyosin in early myogenesis was studied histochemically using monoclonal antibody to alpha tropomyosin and affinity-purified polyclonal antibody to beta tropomyosin. In muscle cells, in both somites and limb buds, the alpha and beta subunits are simultaneously expressed and first appear in the somites at the 30-36 somites. The relatively greater amount of beta than alpha tropomyosin found in early myogenesis is thus likely to result from a higher rate of beta tropomyosin synthesis.

Animals↗
PubMed1989

Tissue-specific transcriptional control of alpha- and beta-tropomyosins in chicken muscle development.

During muscle maturation, isoform switching of contractile proteins to attain the adult phenotype involves both stage-specific and muscle-specific regulatory mechanisms. Chicken pectoralis major (PM) provides an interesting model to study the latter since a specific pattern of tropomyosin (TM) with repression of the beta TM isoform is displayed by the adult PM. The developmental pattern of alpha and beta fast skeletal muscle tropomyosins' (alpha f and beta TM) RNAs was investigated with 3' untranslated region specific probes. In PM, the beta TM messenger ceased to accumulate after hatching through a transcriptional control, as shown by run-on assays, so that, at Day 8 ex ovo, no beta TM mRNA was detected. In this same muscle, in parallel with the disappearance of the beta TM mRNA, there was a boost in the accumulation of the alpha f TM mRNA. In the leg muscles, following hatching, there was only a moderate increase in the level of the alpha f TM mRNA, together with a slight decrease in the accumulation of the beta TM mRNA. Taken together, these results show that chicken muscle maturation involves tissue-specific transcriptional control of tropomyosin genes and could suggest a possible coordinate regulation of the two genes.

Animals↗
PubMed1988

Evidence for distinct phosphorylatable myosin light chains in avian heart and slow skeletal muscle.

In mammalian organisms the regulatory or phosphorylatable myosin light chains in heart and slow skeletal muscle have been shown to be identical and presumable constitute the product of a single gene. We analyzed the expression of the avian cardiac myosin light chain (MLC) 2-A in heart and slow skeletal muscle by a combination of experimental approaches, e.g., two-dimensional gel electrophoresis of the protein and hybridization of mRNA to specific MLC 2-A sequences cloned from chicken. The investigations have indicated that, unlike in mammals, in avian organisms the phosphorylatable myosin light chains from heart and slow skeletal muscle are distinct proteins and therefore products of different genes. The expression of MLC 2-A is restricted to the myocardium and no evidence was found that it is shared with slow skeletal muscle.

Animals↗
PubMed1988

[Molecular strategies involved in the control of gene expression during differentiation of muscle cells].

The initial step in muscle formation is the fusion of undifferentiated myoblasts into multinucleated myotubes which synthesize the specific proteins of the muscle. During this transition a whole series of genes are turned on. A simple explanation for this activation process is that each gene contains a structure which is common to all genes of the family and which is recognized by (a) specific factor(s). Experimental evidence supporting this model is described. Appearance of a new protein may not involve gene activation but be the result of a new mode of splicing as in the case of tropomyosins. Examples have been chosen to illustrate the various strategies used to control gene expression and provide the necessary diversity required during the process of muscle maturation. They include controlled expression of linked genes, coexpression of genes from unrelated phenotypes or alternative splicing of unique genes.

Animals↗
PubMed1988

[Several classes of myoblasts participate in the formation of skeletal muscles in birds].

Our results show that when myoblasts, isolated at different stages during muscle development, were cultured they formed myotubes expressing distinct phenotypes. An early phenotype was expressed by myoblasts isolated from 4-5 day old embryos and this phenotype could be modulated according to the culture conditions, i.e. seeding of the myoblasts as isolated cells or as reaggregated clumps of cells. An intermediate phenotype was expressed by myoblasts isolated from 7-8 day old embryos and it was independent of culture condition. A late phenotype was expressed by myoblasts isolated from embryos older than 9 days. In this case again, it could be modulated by culture conditions but, this time, modulation was brought about by subculturing the cells before they differentiate. These various phenotypes do not result from environmental differences in the culture but reflect the existence of distinct classes of myoblasts present at these different stages. This was demonstrated by isolating homogeneous clones of myoblasts at these stages and by showing that they express the corresponding phenotype.

Animals↗
PubMed1988

[Regulation of the expression of alpha and beta tropomyosin genes during development of the pectoral muscle in the chicken].

Accumulation of mRNAs coding for alpha and beta skeletal tropomyosins was investigated using specific probes and normalized to muscle creatine kinase (M-CK) mRNA by slot-blot assays. In developing pectoralis muscle, the ratio of alpha TM messenger/M-CK remained constant until hatching, at which time the messenger disappeared within a week. However, in the leg, this ratio remained constant until 8 days after hatching, whereafter it decreased progressively to reach 30% in the adult. The alpha TM/M-CK ratios were almost the same in the embryonic leg and pectoralis muscle. After hatching, there was a large increase in pectoralis muscle (x 3 at day +4, x 0 at day +21) whereas, the increase was less pronounced and more progressive in the leg (x 3 at day 21). Run-on assays showed that nuclei isolated from 15-day in ovo leg and pectoralis muscles had similar patterns of muscle specific gene transcription whereas post-hatched pectoralis muscle nuclei were shown to have a higher rate of alpha to beta tropomyosin gene transcription. These data are in accordance with the results obtained for protein analysis of leg and pectoralis muscles and support the notion that changes in the protein pattern of developing muscle can be relevant to coordinate regulation of gene transcription.

Animals↗
PubMed1987

Chick and quail limb bud myoblasts, isolated at different times during muscle development, express stage-specific phenotypes when differentiated in culture.

Evidence is presented which shows that myoblasts, isolated at different stages during chick and quail limb bud development, will form, in culture, myotubes which can be distinguished with a combination of morphological as well as biochemical criteria. Hind limb bud myoblasts isolated from 5-day-old embryos form very short myotubes which synthesize a myosin, the light chains of which are predominantly LC1F and LC2S. Myoblasts isolated from the limb buds of 7-8-day-old embryos form large myotubes which synthesize a myosin the light chains of which are predominantly LC1F, LC2S and LC2F. Myoblasts isolated from the thigh muscle of embryos older than 10 days form large myotubes which synthesize a myosin the light chains of which are predominantly LC1F and LC2F. These results have been confirmed by hybridization of the cellular mRNA with a molecular probe specific for LC2F. These results lead us to suggest the existence of at least two classes of myoblasts which appear at different times during limb bud development. The first class, or 'early' myoblasts, is present in the limb buds of 5-day-old embryos, whereas the second class, or 'late' myoblasts, is present in the muscles of embryos older than 8 days. This result, however, is also compatible with the hypothesis that all muscle cells are the same at all times during development, and that the different phenotypes simply reflect differences in the environmental conditions.

Animals↗
PubMed1986

Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis.

Primary isolates of chick leg muscle myoblasts cultured on hyaluronic acid substrates have been examined by transmission electron microscopy for evidence of myoblast fusion and subsequent differentiation. Even though these cells form close contacts, no evidence of multinucleated myotubes is found in these cultures. Two-dimensional SDS-polyacrylamide gel electrophoresis shows that the muscle macromolecular biosynthetic program is not initiated in these hyaluronic acid fusion-blocked cells. Further, these fusion-blocked myoblasts continue replicating while cultured on hyaluronic acid surfaces. The inhibition of both fusion and the myogenic expressional program is reversed by replating these myoblasts onto a denatured collagen (gelatin) substrate; both the synthesis of muscle-specific proteins and the formation of multinucleated myotubes are observed when these subcultured cells are introduced onto gelatin substrates. These observations indicate that the hyaluronic acid inhibition of fusion is not permanent and is manifested in a way different from other fusion blockers in that hyaluronic acid inhibits both fusion and the myogenic expressional program.

Actin Cytoskeleton↗