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D Yaffe

Publications and source records attributed to D Yaffe.

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

Characterization and cell type distribution of a novel, major transcript of the Duchenne muscular dystrophy gene.

Previously we identified a novel 6.5 kb mRNA transcribed from the Duchenne muscular dystrophy (DMD) gene. This mRNA differs in coding content and tissue distribution from the known muscle type and brain type 14 kb DMD mRNAs which code for dystrophin. The novel transcript shares with dystrophin most of the sequence coding for the cysteine-rich and C-terminal domains. Here we used cDNA cloning to identify the divergence point between the common region and the sequence unique to the novel mRNA at the 5' end of the sequence encoding the cysteine-rich domain of dystrophin. This unique sequence containing the translation initiation site is located in a new exon in the intron between exons 62 and 63 of the dystrophin gene. Using probes containing RNA sequences specific to the novel mRNA, we investigated the expression of this mRNA in various tissues and cell types. The study reveals that this mRNA is the main DMD gene product detectable in a variety of nonmuscle tissues including brain cells. The amount of this mRNA in some tissues is comparable to the amount of dystrophin mRNA in the muscle. The expression of the 6.5 kb mRNA is down-regulated during differentiation of myogenic cells; it is present in small amounts in proliferating myoblasts but is undetected in differentiated muscle cultures depleted of mononucleated cells.

Animals↗
PubMed1992

Multiple products of the Duchenne muscular dystrophy gene.

The gene which is defective in Duchenne Muscular Dystrophy (DMD) extends over 2300 kb of the X chromosome. Its product in the muscle is a 14 kb mRNA encoding a 427 kd rod-shaped protein called dystrophin. A 14 kb transcript encoding a very similar isoform of dystrophin is produced in the brain. The brain 14 kb mRNA is transcribed from the same gene but controlled by a different promoter, located at least 75 kb upstream from the muscle dystrophin promoter. The regulation of these promoters is very stringently controlled. The muscle-type but not the brain-type dystrophin mRNA is found in cloned skeletal muscle cells and its presence is correlated with the appearance of multinucleated fibers. The brain type is expressed in neurons, while in glia cells the muscle-type promoter is active. A third DMD gene transcript which is only 6.5 kb long has been identified. It contains the sequence coding for the C-terminal domain and the cysteine-rich domain of dystrophin but not the large region encoding the spectrin-like repeats and the N-terminal domain. The cell type distribution of this transcript is also very different from that of the two 14 kb mRNA isoforms. It is the major product of the DMD gene in many nonmuscle tissues including brain. Using monoclonal antibodies we have identified a 77 Kd protein which seems to be the translation product of this mRNA. As expected from the distribution of the 6.5 Kb mRNA, this protein is the major DMD gene product detectable in brain and many other nonmuscle tissues; it is undetectable in skeletal muscle but is present in the heart and stomach (as is the 6.5 Kb mRNA).

Base Sequence↗
PubMed1991

Cowper's glands duct: radiographic findings.

Lesions of Cowper's glands duct are uncommon findings in a urethrogram. Three types of anomaly are described in 11 patients. The most frequent type is a slightly dilated duct, which is usually asymptomatic, with or without associated urethral pathology. The other less common types are perforate Cowper's duct and retention cyst, which are usually symptomatic and not associated with other urethral pathologies.

Adult↗
PubMed1991

Brain-type and muscle-type promoters of the dystrophin gene differ greatly in structure.

The promoter of the 14 kb mRNA encoding the brain isoform of dystrophin in the mouse has been isolated and partially characterized. Unlike the promoter of the muscle dystrophin isoform, it does not contain a TATA box or other consensus sequences characteristic of the proximal region upstream of the cap sites of eukaryotic genes. Yet, it has a major initiation of transcription start site located 266 bp upstream from the first ATG which is in frame with the dystrophin coding sequence. The 5' untranslated region contains nine additional ATG triplets which are not in-frame with the coding sequence or are followed by stop codons. A DNA fragment extending from bp -1149 to +11 is sufficient to activate a reporter gene lacking a promoter in transfected neuroblastoma cells.

Animals↗
PubMed1991

Mapping of dystrophin brain promoter: a deletion of this region is compatible with normal intellect.

Using a mouse genomic fragment containing the brain-specific promoter region of the dystrophin gene, we have located the brain promoter 75-300 kb proximal of the muscle promoter. Within our DMD-families we detected a patient who lacks both the brain-specific and muscle-specific promoter sequences. The normal intellectual capabilities of the patient argue against an indispensable role of the brain-specific first exon in mental functioning. The possibility exists that a NH2-terminally truncated dystrophin has taken over the function of the normal dystrophins in brain and/or muscle.

Adult↗
PubMed1990

A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution.

A novel transcript of the Duchenne muscular dystrophy gene has been identified. This 6.5 kb mRNA contains sequences from the 3' untranslated region of dystrophin mRNA and from the regions coding for the C-terminal and the cysteine-rich domains. However, probes for the regions encoding the spectrin-like repeats and the actin-binding domain, as well as probes for the first exons of the muscle- and brain-type dystrophin mRNA, did not hybridize with this new mRNA. Significant amounts of the 6.5 kb mRNA were found in a variety of non-muscle tissues, such as liver, testis, lung and kidney, but not in skeletal muscle. The abundance of this mRNA in the brain is at least as high as that of the previously described 14 kb brain-type dystrophin mRNA.

Animals↗
PubMed1990

Specificity of expression of the muscle and brain dystrophin gene promoters in muscle and brain cells.

The gene that is defective in Duchenne and Becker muscular dystrophies is expressed in the muscle and brain. However, the 5' ends of the 14 kb mRNA in these tissues are derived from two different exons, indicating the involvement of at least two promoters in the regulation of the cell-type and developmental specificities of expression of this gene. In the study presented here, we used the polymerase chain reaction and RNAase protection methods and various cell cultures to investigate the specificities of expression of these promoters. The results indicate a very stringent control of expression of the two promoters. In cloned rat myogenic cells, only the muscle-type dystrophin transcript was detected, and its presence was correlated with the formation of multinucleated fibers. In neuronal cell cultures, the brain-type transcript was detected. However, glial cell cultures expressed the muscle transcript only. Some cell lines derived from brain cells expressed both isoforms.

Animals↗
PubMed1990

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes.

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.

Animals↗
PubMed1990

The amount of the endogenous and exogenous skeletal muscle actin mRNA in the heart of transgenic mice is affected by the genotype of the cardiac actin gene.

Both skeletal muscle and cardiac actins are co-expressed in the newborn heart. However, the amount of the skeletal muscle actin and its mRNA rapidly decreases during early development and the cardiac actin predominates in the adult heart. In BALB/c and DBA mice there is a mutation in the cardiac actin gene which is associated with decreased levels of cardiac actin mRNA and high levels of the skeletal muscle actin transcript in the adult heart. To examine the possibility that the amount of cardiac actin gene product modulates the expression of the skeletal muscle actin gene in the heart, transgenic mice carrying a tagged skeletal muscle actin gene were produced, and the expression of the endogenous and endogenous and exogenous actin gene was analyzed in offspring carrying different combinations of the cardiac actin alleles. It was found that both the endogenous and exogenous skeletal muscle actin genes were expressed at low levels in the heart of adult mice homozygous for the wild-type cardiac actin gene allele, at abnormally high levels in mice homozygous for the mutated cardiac actin allele, and at intermediate levels in heterozygous mice. This shows that the level of expression of the cardiac actin gene has a trans effect on the expression of the skeletal muscle actin gene.

Actins↗
PubMed1989

Duchenne muscular dystrophy gene product is not identical in muscle and brain.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting in progressive degeneration of the muscle. It affects about 1 in 3,500 male children. Becker's muscular dystrophy is a less severe disease allelic to DMD. Some 30% of DMD patients suffer from various degrees of mental retardation. The giant DMD gene spans about 2,000 kilobases and codes for a 14-kilobase messenger RNA and a protein of molecular weight 427,000. DMD mRNA is most abundant in skeletal and cardiac muscle and less so in smooth muscle. We reported that the expression of the gene is developmentally regulated during the differentiation of primary muscle cultures and in myogenic cell lines in a way similar to the expression of muscle-specific genes such as myosin light chain 2 and skeletal muscle actin. Similar results have been obtained with human primary myogenic cells. Significant levels of DMD mRNA are found in brain tissue. Here we show that the transcript of the DMD gene and the amino terminal of the encoded protein differ in brain and muscle. The 5' ends of these mRNA species are derived from different exons. The results suggest that the two mRNA types are transcribed from different promoters.

Animals↗
PubMed1989

Regulated expression of muscle-specific genes introduced into mouse embryonal stem cells: inverse correlation with DNA methylation.

Pluripotent embryonal stem cell lines (ES) were isolated from cultured normal mouse blastocysts. These cells retained their capacity to differentiate into a great variety of cell types in cell cultures or in tumors formed after subcutaneous injection of the cells into nude mice. A chimeric actin/globin gene containing about two-thirds of the rat skeletal muscle actin gene and 730 bp of its 5' flanking region fused to the 3' end of the human embryonic epsilon-globin gene, was inserted into a plasmid containing a neomycin resistance gene (neor) whose transcription is regulated by the SV40 early control elements. The prokaryotic vector DNA sequences of this plasmid (pAG-Neo) were deleted and the two linked genes were introduced into the ES cells by electroporation. G418-resistant clones were isolated, amplified and injected subcutaneously into nude mice. From the teratocarcinoma-like tumors which developed we isolated myogenic as well as nonmyogenic cell lines. In cell lines derived from three independent transfected ES clones, expression of the actin/globin gene was developmentally regulated in myogenic cells. In contrast, in a number of experiments in which the actin/globin gene or other muscle-specific genes were introduced into the ES cells without the removal of the pBR sequences, no expression could be detected at any stage. Moreover, in the differentiated lines derived from these clones, G418 resistance was lost, and no neor transcripts could be detected. Southern-blot analysis of MSPI- or HpaII-digested DNA revealed extensive methylation in the clones that did not express the foreign DNA, whereas no significant methylation of the inserted DNA was observed in clones which expressed the transfected genes. Examination of the DNA extracted from transgenic mice carrying the same actin/globin gene revealed an inverse correlation between methylation of the exogenous gene and its potential to be expressed in the transgenic strain. However, no tissue-specific differences in methylation, related to the tissue specificity of expression of the exogenous gene, could be detected in these experiments. These results suggest that the process of methylation reported here is causally related to constitutive inactivation of the exogenous genes.

Actins↗
PubMed1988

Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain.

Duchenne muscular dystrophy (DMD), a sex-linked degenerative disorder of the muscle, is one of the most common lethal genetic diseases in man. It affects about one male in 3,500, with an estimated one-third of cases being caused by new mutations. A less severe disease, Becker's muscular dystrophy (BMD), maps to the same chromosomal locus and is most probably an allelic form of DMD. Both diseases are sometimes associated with various degrees of mental retardation; the molecular basis of these phenotypes is unknown (for review, see ref. 1). The giant DMD gene spans approximately 2,000 kilobases (kb) (0.05% of the human genome) and encodes a 14-kb mRNA. The tissue-specificity of its expression has not been precisely determined. Monaco et al., using Northern blots, reported expression of the gene in human fetal skeletal muscle and small intestine but not in human fetal brain, or in human cultured myoblasts and transformed B and T cells. More recently, expression was detected in mouse skeletal and cardiac muscle, but not in mouse brain. Here we show, using a ribonuclease protection assay, that the DMD gene is developmentally regulated in rat and mouse myogenic cell cultures, and that it is expressed in rat and mouse striated muscle, in mouse smooth muscle and in rat, mouse and rabbit brain. We could not detect transcripts in other non-muscle tissues.

Amino Acid Sequence↗
PubMed1987

Expression in transgenic mice of two genes of different tissue specificity integrated into a single chromosomal site.

Transgenic mice were used to study the expression of pairs of genes with distinctly different tissue specificities, covalently linked and integrated into the same chromosomal site. A transgenic strain carrying, in close proximity and in the same orientation, the rat fast skeletal muscle myosin light-chain 2 (MLC2) gene and the mouse rearranged immunoglobulin kappa light-chain gene expressed the immunoglobulin gene specifically in the lymphoid tissues, whereas rat MLC2 transcripts were found in skeletal muscle but not in the spleen or the other tissues that were tested. In another transgenic strain, carrying the rat MLC2 gene and a modified rat skeletal muscle actin gene (actin-globin chimeric gene), transcripts of the rat MLC2 gene were detected in skeletal muscle only, whereas the actin-globin transcripts were detected in skeletal muscle as well as in the heart. Moreover, the expression of the chimeric gene was also developmentally regulated. Expression was higher in cardiac muscle than in the skeletal muscle of neonatal mice, whereas expression was higher in skeletal muscle in adult mice. This pattern is consistent with the regulation of the expression of the endogenous skeletal muscle actin gene. Thus, in those transgenic strains that expressed both genes, each gene retained its tissue specificity, in spite of their close proximity. These results indicate a high degree of autonomy of the control elements included in the cloned genomic DNA fragment and demonstrate that a single chromosomal site can be permissive for the proper expression of two genes with different tissue specificities.

Actins↗
PubMed1986

Muscle-specific activation of a methylated chimeric actin gene.

To understand how DNA methylation affects tissue-specific activation of genes, we have transfected in vitro methylated alpha-actin (skeletal) constructs into fibroblasts, which do not produce endogenous alpha-actin, and into a myogenic line, which is inducible for alpha-actin expression. Although methylation significantly inhibits the expression of these constructs in fibroblasts, it does not in myoblasts. The methylation pattern of the introduced methylated genes reveals specific demethylations in the transfected molecules in myoblasts but not in fibroblasts, and it precisely mimics the methylation pattern found in myoblasts in vivo.

Actins↗
PubMed1985

Highly conserved sequences in the 3' untranslated region of mRNAs coding for homologous proteins in distantly related species.

Comparison of the nucleotide sequence of mRNAs coding for several vertebrate actins revealed a high degree of sequence homology in the 3' untranslated region (3' UTR) between those mRNAs coding for homologous (isotypic) actins in different organisms but not between mRNAs coding for very similar isoforms differing in their function or tissue specificity. A similar pattern of sequence conservation in the 3' UTR is also found in several other genes. Furthermore, while there is a great variation in the size of the 3' UTR of mRNAs coding for different proteins, mRNA coding for isotypic proteins in distantly related organisms often have 3' UTR of similar size. The data suggest that the 3' UTR may play an important role in the regulation of expression of at least some genes at the transcriptional or posttranscriptional level.

Actins↗
PubMed1985

Developmentally regulated expression of a chicken muscle-specific gene in stably transfected rat myogenic cells.

To test the evolutionary conservation of DNA sequences specifying the developmentally regulated expression of the skeletal muscle actin gene, a recombinant plasmid containing the chicken skeletal muscle actin gene was introduced into rat myogenic cells. In a significant number of isolated clones, the accumulation of chicken actin mRNA increased greatly during differentiation. To test the expression in myogenic cells of a gene that is normally expressed during terminal differentiation of another tissue, rat myogenic cells were transfected with a mouse/human beta-globin chimeric gene. A decrease by a factor of 2-3 in the amount of globin mRNA during differentiation was observed in most clones in which the gene was expressed. The results indicate the conservation of the muscle-specific regulatory DNA sequences for more than 300 Myr.

Actins↗