Recombinant DNA clones containing sequences for two different types of myosin heavy chain (HC) genes from chicken embryonic skeletal muscle were constructed and analyzed. Specificity of the clones for myosin HC was demonstrated by hybrid-arrested translation, by hybridization to a 7.0-kb mRNA, and by comparison of DNA sequences with known amino acid sequences of rabbit skeletal muscle myosin HC. Restriction enzyme and electron-microscopic heteroduplex analysis showed the presence of two distinct but homologous cDNA sequences. Hybrid melting curves indicated that both types of sequences represent fast myosin HC sequences.Skeletal muscle myosins are known to exist in multiple polymorphic forms that result from the expression of different myosin heavy chain (HC) and light chain genes (1-3). Additional myosin types are present in cardiac muscle (4, 5) and in nonmuscle tissue (6). If one considers these known genes, the possibility ofpseudogenes, and perhaps additional genes for which specific products have not yet been characterized, the myosin heavy chain gene family may be quite large. A minimal estimate of three myosin HC genes has been derived from screening of a rat gene library with cloned rat myosin HC cDNA (7).The expression of skeletal muscle myosin follows a characteristic developmental pattern (2, 3, 8-10). Examination of the gene structure and ofthe organization ofthe myosin gene family and analysis of transcription of the different molecular forms may reveal how these genes are regulated during development and how their expression is altered by physiologic stimuli.We have partially purified and separated mRNAs for fast and for slow myosin HCs from chicken embryonic skeletal muscle (11). However, each ofthese mRNA preparations probably contains more than one molecular form. To study the expression of individual myosin HC genes more specifically, we used bacterial plasmids to clone cDNA sequences of myosin HC mRNA isolated from embryonic chicken leg muscle. Restriction endonuclease mapping, electron-microscopic heteroduplex analysis, and hybrid melting curves indicate that these clones contain sequences for two distinct fast myosin HC genes.MATERIALS AND METHODS Preparation of RNA. Polysomes were isolated from 14-dayold embryonic chicken leg muscles as described (12). Poly(A)-containing RNA was purified from the polysome pellets by extraction with phenoVchloroform/isoamyl alcohol and by two cycles of chromatography on oligo(dT)-cellulose [T3, Collaborative Research (Waltham, MA)] (13). The poly(A)-containing RNA was sedimented twice on 10-30% sucrose gradients in a Beckman SW41 rotor at 28,000 rpm for 16 hr at 20TC, and the fractions containing 26-32S RNA were pooled.For hybrid-arrested translation and reverse Southern hybridization, polysomal RNA was isolated from 14-day-old embryonic chicken leg muscle polysomes purified on 1.8-2.5 M discontinuous sucrose gradients (11).Construction and Screening of cDNA Clones. The synthesis of single-stranded (ss) and double-stranded (ds) cDNA was accomplishe...
The transcription and processing of mitochondrial 21S rRNA in a petite strain of Saccharomyces cerevisiae has been examined by electron microscopic analysis of R-loop hybrids and by hybridization of labeled mitochondrial DNA probes to RNA transferred to diazobenzyloxymethyl paper. We have shown the presence of a large [5.1-to 5.4-kilobase (kb)J transcript that appears to be a precursor of mitochondrial 21S rRNA. This transcript contains sequences homologous to those of the mature 21S rRNA, to the intervening sequence present in the gene, and to additional sequences at the 3' end of the molecule. Our data suggest that this precursor of 21S rRNA is processed in two steps. The intron sequence is usually excised first, followed by removal of the extra 3' sequences. In some cases, however, the 3' extension is first removed and the intron sequence is then excised. Both pathways appear to lead to formation of the 3.1-kb mature 21S rRNA and a stable 1.2-kb intron transcript. Similar results were obtained with grande MH41-7B mitochondrial RNA by RNA transfer hybridization. We have also observed a number of additional transcripts that may be normal processing intermediates or may result from faulty cleavage-ligation during excision of the intervening sequence. The 70--to 75-kilobase (kb) yeast mitochondrial genome and its products have been analyzed extensively. Detailed genetic (e.g., refs. 1 and 2) and restriction endonuclease maps have been derived (e.g., refs. 3-6), regions of the DNA sequence have been determined (e.g., refs. 7-9), and a number of the transcripts have been characterized and mapped (10-15). Yeast mitochondrial DNA (mtDNA) specifies mitochondrial 14S and 21S rRNA, about 25 tRNAs, and seven to nine polypeptides (for review, see ref. 16). Although these gene products account for at most 20-30% of a single-strand DNA equivalent, more than 60% of the mitochondrial genome is transcribed (17). Analysis of mitochondrial transcripts from grande and petite strains by gel electrophoresis has shown that their aggregate molecular weight exceeds the coding capacity of the genome (11,12). Similarly, transcript mapping with petite yeast strains indicates that multiple RNA species are specified by individual regions of the mitochondrial genome (11,13). Therefore, large regions of the genome appear to be transcribed, and the products are then processed into mature RNA species. Furthermore, intervening sequences have been demonstrated in the cytochrome b (18, 19) and the 21S rRNA (20-22) cistrons and possibly the OXI 3 region (unpublished results). Transcripts derived from these regions thus require extensive processing. Characterization of RNA processing pathways would appear to be essential for an understanding of the biogenesis of mitochondria.In this study, we have focused on the analysis of transcripts derived from the 21S rRNA cistron. This gene contains a 1.2-kb intervening sequence (20)(21)(22) (11,13).The Fl1 mitochondrial genome has been extensively characterized both genetically and physically (23,25...
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