Complete amino-acid sequence of subfragment-2 in adult chicken skeletal muscle myosin

Although the complete amino-acid sequence of the short subfragment-2 (short S-2) and the partial sequence of the hinge region derived from adult chicken skeletal muscle myosin have been reported previously, the sequence of the N-terminal portion of subfragment-2 (S-2) and the connective portion between the above two regions could not be determined. In this study, the amino-acid sequence of these undetermined portions were completely sequenced. Furthermore, overlaps of cyanogen bromide (CNBr) peptides in the hinge region were also isolated and sequenced. Peptides obtained by hydrolysis with dilute formic acid and by digestion with lysyl endopeptidase of S-2 were purified and sequenced. These results established the complete amino-acid sequence of S-2 composed of 429 amino-acid residues. This sequence of adult chicken skeletal muscle myosin was compared with that of chicken embryonic skeletal muscle, chicken gizzard muscle and rabbit cardiac muscle myosin (alpha-myosin heavy chain) and shows degrees of 96%, 38% and 84% sequence identities, respectively. The frequency with which hydrophobic residues are present at position "a" in seven-residues repeats of the hinge region was markedly reduced when compared to the short S-2 sequence of the chicken skeletal muscle myosin.     

Complete nucleotide sequence of a cloned chicken alpha-globin cDNA.

Chicken globin double-stranded cDNA was synthesised from anaemic adult reticulocyte alpha- and beta-globin mRNA and ligated into the Hind III site of pBR322 using synthetic Hind III decamers. Transformation of E. coli x1776 resulted in the production of a number of alpha- and beta-cDNA clones. One of the alpha-type clones (pCG alpha-8) was fully sequenced and found to code for neither alpha A- nor alpha D-globin. Partial sequencing of the other alpha-cDNA clones indicates that they are all of the same type.     

Amino acid sequence of rabbit skeletal muscle alpha-tropomyosin. The COOH-terminal half (residues 142 to 284)

Amino acid sequence analysis of the large cyanogen bromide fragment (residues 142 to 281) derived from the COOH-terminal half of the mixed tropomyosin population of rabbit skeletal muscle has been carried out. The isolation and sequence analysis of peptides derived from chymotryptic digests and from tryptic digests of the maleylated fragment permitted the alignment of the complete sequence except for the assignment of acids or amides at residues 142, 144, and 145. Selected peptides from a Myxobacter 495 alpha-lytic protease digest have confirmed certain overlaps. Based on previously published data the sequence can be extended to residue 284, the COOH-terminal end of the protein. In fourteen positions, amino acid substitutions have been observed. In one of these (residue 199) the sequence evidence indicates a minimum of four different polypeptide chains in the mixed tropomyosin population. The assignment of particular amino acid residues to these positions for the major alpha-component of rabbit skeletal tropomyosin has been based on the relative recoveries of peptides containing different residues in these positions.     

A new single nucleotide polymorphism in the ryanodine gene of chicken skeletal muscle

Some genes affect meat quality in chickens. We looked for polymorphisms in the Gallus gallus α-RyR gene (homologous to RyR 1) that could be associated with PSE (pale, soft and exudative) meat. Because RyR genes are over 100,000 bp long and code for proteins with about 5000 amino acids, primers were designed to amplify a fragment of hotspot region 2, a region with a high density of mutations in other species. Total blood DNA was extracted from 50 birds, 25 that had PSE meat and 25 normal chickens. The DNA samples were amplified by PCR, cloned, sequenced, and used to identify single nucleotide polymorphisms (SNPs). The amplified fragment of α-RyR was 604 nucleotides in length; 181 nucleotides were similar to two exons from a hypothetical turkey cDNA sequence for α-RyR. A non-synonymous nucleotide substitution (G/A) was identified in at least one of the three sequenced clones obtained from nine animals, six PSE (HAL+) birds and three normal (HAL-) birds; they were heterozygous for this mutation. This SNP causes a change from Val to Met in the α-RYR protein. Since the frequencies of this SNP were not significantly different in the PSE versus normal chickens, it appears that this mutation (in heterozygosity) does not alter the structure or function of the muscle protein, making it an inappropriate candidate as a genetic marker for PSE meat.     

Increased wheel-running activity in the genetically skeletal muscle fast-twitch fiber-dominant rats.

The purpose of the present study was to investigate whether genetic differences in muscle histochemical characteristics were related to the voluntary wheel-running activity level by using genetically fast-twitch fiber-dominant rats (FFDR) and control rats (CR). The rats were divided into four groups; sedentary CR (Sed-CR), wheel-running CR (WR-CR), sedentary FFDR (Sed-FFDR), and wheel-running FFDR (WR-FFDR). Wheel access was started at age 9 wk and lasted for 7 days. The FFDR showed a lower percentage of type I fibers of the deep portion of gastrocnemius and soleus muscles and a higher percentage of both type IIX fibers of the gastrocnemius muscle and type IIA fibers of the soleus muscle compared with CR. A higher capillary density and smaller fiber cross-sectional area were also observed in FFDR. The daily running distance in WR-FFDR was higher than in WR-CR for each 7 days. The total running distance for 7 days in WR-FFDR was 3.2-fold higher than in WR-CR. On day 7 of the 7-day test, the total number of active 1-min intervals for 24 h, the average rpm when they were active, and the maximum rpm for any single 1-min period in the WR-FFDR were significantly higher than in the WR-CR (1.5-, 2.9-, and 2.0-fold, respectively). These results suggest that mechanical or physiological muscle characteristics may thus affect the wheel-running activity level.     

Calcium currents in a fast-twitch skeletal muscle of the rat

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.     

Complete nucleotide sequence of the chicken alpha A-crystallin gene and its 5' flanking region

The chicken alpha A-crystallin gene and 2.6 kb of its 5' flanking sequence have been isolated and characterized by electron microscopy and sequencing. The structural gene is 4.5 kb long and contains two introns, each approx. 1 kb in length. The first intron divides codons 63 and 64, and the second intron divides codons 104 and 105, as in rodents. There is little indication that the insert exon of rodents (an alternatively spliced sequence) is present in complete form in the chicken alpha A-crystallin gene; small stretches of similarity to this sequence were found throughout the gene. The 5' flanking sequence of the chicken alpha A-crystallin gene shows considerable sequence similarity with other mammalian alpha B-crystallin genes. In addition, one consensus sequence (GCAGCATGCCCTCCTAG) present in the 5' flanking region of the chicken alpha A-crystallin gene was found in the 5' flanking region of most reported crystallin genes.