We have used a myosin heavy chain gene fragment to probe two chicken genomic libraries.The fragment, derived from a gene expressed in the fast-white fibers of adult chickens, contains 1400 bases upstream from the translational start site and 1600 bases downstream from the initiation codon.Thirty-one unique nonoverlapping clones were isolated.All of the genes showed homologies in the nucleotide sequences which code for the globular head portion of the myosin protein while no extensive homologies were detected in the 5"flanking sequences.The relationships between the genes were studied using oligomeric sequences as probes.The hybridization patterns showed that seven of the genes fall within a well defined subgroup.The exon containing a domain of the nucleotide (ATP) binding site was sequenced for all seven of these genes and shown to be, except for 2 nucleotides in one of the genes, completely conserved.The lack of sequence conservation in the 5"nontranslated portions of the genes was exploited in the preparation of transcript-specific probes.We have used these probes to show that two of the isoforms are expressed in a tissue-specific and developmental stage-specific manner in the chicken.
The complete sequence of an embryonic chicken myosin heavy chain has been determined. Introns and exons were identified by comparison with the corresponding cDNA. The cDNA contains 5,962 bases, of which 85 bases constitute the poly(A) tail. The cDNA represents the entire mRNA transcript, except for 90 bases at the 5'-coding terminus and 101 bases of the 5'-untranslated region. The gene's coding region is split by 37 introns; two additional introns split the 101 base pairs which make up the 5'-untranslated region. The complete gene is approximately 23,000 base pairs and encodes a protein whose molecular weight is 222,559 and consists of 1,940 amino acids. Analysis of the protein and comparison with other myosin sequences reveal that certain regions have been conserved; those amino acids which have been postulated to participate in the ATPase and actin-binding activities of the molecule are highly homologous. These comparisons have allowed the identification of isolated regions within the myosin heavy chain that appear to be essential for the molecule's function.
Abstract The sequences encoding the 5'-ends of three chicken fast-white myosin heavy chain (MHC) genes have been determined. When compared with the sequences of two other MHC genes it is apparent that both the exon and intron positions are conserved. All exon sequences are highly conserved; there is absolute amino acid conservation in the second and third exons. In addition, while the first and third introns diverge among the genes, the second intron is highly conserved between the five. This intron contains a 24-bp sequence that is repeated twice in one of the introns and once in the other four. Analyses indicate that this sequence, which is partially homologous to 7SL RNA, appears to be largely restricted to the MHC gene family. Analysis of the 5'-flanking sequences show that while small homologies are present between some of the genes, they have extensively diverged in this region.
Two complete myosin heavy chain genes were isolated from chicken genomic libraries, and shown to code for fast-white isoforms. Isoform specific probes were developed from the 5' nontranslated regions of the two genes and used to identify the developmental stages at which each of the genes are expressed. One of the genes is transcribed in the embryo and the other only in the adult. The 5' flanking regions of the two genes were sequenced along with the first three exons. The 5' untranslated sequences in both genes are not contiguous, one intron is present in the adult gene while the embryonic gene contains two. The promoters of both genes contain the conserved CAAT and TATA box elements observed in other eucaryotic genes. A computer assisted comparison was performed on the two genes at the nucleotide and amino acid levels. No homology could be detected in the 5' flanking regions of the genes except in and around the CAAT and TATA elements, however, structural sequences at the 5' ends were highly conserved as well as the position of the first three introns. The amino acids in and around the ATP binding site are completely conserved between the two isoforms.
Recently we have isolated a large number of chicken myosin or myosin-like heavy chain genes. Seven of these genes were placed into a subset based upon their hybridization patterns. In the present study, the sequence of the 5' end of one of the myosin heavy chain (MHC) genes, N127, was determined and compared with the 5' end sequences of the other six MHC genes in the subset. The comparison revealed that the three exons encoding the amino termini of the protein are highly conserved. The sequence analysis shows that a localized correction event occurred in and around a domain of the nucleotide-binding site, as the exon encoding this site and the preceding intron are very highly conserved among the seven genes. The sequence of the promoter and 5'-untranslated region of N127 is presented. The analogous regions for N124 and N125 have now been sequenced and are also presented. As is the case for all the other known MHC genes, the 5'-untranslated regions are split by large introns. The promoter and 5'-untranslated regions are compared with two previously characterized chicken MHC genes (N116 and N118) to determine the sequence similarities and differences that might underlie the differential expression of the family's members. To confirm and extend previously published results of the expression of these genes, transcript-specific probes generated from the 5' region of six of the seven genes were used to determine in which muscle(s) the corresponding mRNAs were present. The data show that despite the very close structural homologies, each of the genes for which a unique probe could be prepared exhibits a unique pattern of expression.
