ABSTRACT
Differences in molecular weights and partial amino acid sequences of connectin(titin) were determined for cattle, pig and chicken skeletal muscles. Peptide mapping analysis results differed ...according to animal species. Amino acid sequences deduced from partial nucleotide sequences of connectin also differed according to animal species at immunoglobulin‐like (Ig) and fibronectin type 3 (FN3) domains. In chicken, the molecular weight of connectin from leg muscles was higher than that from pectoral muscles. Differences in meat texture and conditioning may relate to connectin and extent of its breakdown.
Nucleotide sequences which included the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from equine skeletal muscles. The deduced amino acid sequences were ...1937, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. No MyHC-2b isoform was amplified from the equine muscle cDNA except for one pseudogene fragment. One nucleotide was inserted in the coding region of the equine pseudogene product, a minute amount of which was expressed in the skeletal muscle. The 596 bp sequence of the equine MyHC pseudogene was categorized into the MyHC-2b genes on the phylogenetic tree of the mammalian MyHC genes. These results suggest that an ancestral MyHC-2b gene had lost its function and changed to a pseudogene during the course of horse history. The MyHC genes in some ungulates were analyzed through the PCR amplifications using the MyHC isoform-specific primers to confirm the presence of the MyHC-2b and -2x genes. The exon coding the 3′ untranslated region of the MyHC-2x was successfully amplified from the all ungulates examined; however, that of the MyHC-2b gene was amplified only from horses, pigs and lesser mouse deer. The PCR analyses from rhinoceros, sika deer, moose, giraffes, water buffalo, bovine, Japanese serow and sheep genes implied the absence of the MyHC-2b-specific sequence in their genomes. These results suggest that the MyHC-2b gene independently lost its function in some ungulate species.
Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was ...recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.
In this study, 10 troponin T isoforms from adult porcine skeletal muscle messenger RNA were clarifed. These were eight fast- and two slow-type isoforms. Fast-type isoforms had three and two variable ...exons in the N-terminal and the C-terminal region respectively. Slow-type isoforms had one variable exon in the N-terminal region.
A novel repeated sequence specific to male cattle was identified and named S4. S4 is a highly repetitive sequence and is a 1.5 kb repeating unit that contains various internal repeated sequences. ...FISH analysis showed that S4 is localized on the whole long arm and the proximal region of the short arm of the Y chromosome. We found that a PCR primer set for S4 amplified a male-specific 178 bp product in addition to a 145 bp product common to both male and female cells. Although the origin of the 145 bp product is unknown, it acts as a positive internal control in practical embryo sexing. Due to the high copy number of S4, PCR required only 0.5 pg purified DNA for accurate amplification. This made it possible to reduce the amount of biopsy sample required for embryo sexing and thus result in less damage to embryos manipulated. These studies indicate that embryo sexing based on the S4 sequence is accurate and sensitive.
To investigate the roles played by MyoD in the terminal differentiation of satellite cell‐derived myoblasts, the effect of antisense inhibition of MyoD expression was examined in bovine adult ...myoblast culture, in which inhibition treatment was limited to the terminal differentiation phase. MyoD antisense oligonucleotide DNA (AS‐mD) suppressed the formation of multinucleated myotubes in the cell culture. Myotube formation was suppressed even when AS‐mD treatment was limited to the period preceding the onset of myotube formation. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis revealed that treatment with AS‐mD suppressed the expression of myosin heavy chain embryonic isoform and troponin T isoforms at 4 days after the induction of differentiation. AS‐mD also suppressed the expression of MRF4, but did not alter the expression of either Myf5 or myogenin, in contrast to previous results using mouse cells possessing MyoD(–/–) genetic background. These findings suggest that MyoD controls myogenesis but not Myf5 or myogenin mRNA expression during the terminal differentiation phase. Furthermore, among the α4, α5, α6, and α7 integrins, α4, α5, and α7 integrin expression was suppressed by AS‐mD treatment, in parallel with the suppression of myotube formation, which suggests that MyoD controls myotube formation by regulating the expression of α4, α5, and α7 integrins.
We describe a rapid, reliable method for the sexing of domestic pigs (
Sus scrofa domestica) by amplification of Y-chromosome specific sequences in male genomic DNA using the polymerase chain ...reaction (PCR). Oligonucleotide primers were selected from a conserved sequence motif, the HMG box, in the sequence of porcine
Sry. These primers permit amplification of a defined 146 by fragment only from male-specific genomic DNA. As a control, autosomal-specific primers were also used in the PCR reactions, recognising a sequence from a porcine homeobox gene that is amplifiable in genomic DNA from both males and females. This sensitive method will find application in the sexing of biopsied, pre-implantation embryos and foetuses used for the derivation of embryonic cell lines and germ-cell derivatives, for use in the new biotechnologies; and in the analysis of male-specific sequences in intersex pigs.
Myosin is a major structural protein of the thick filament of the sarcomere. It constitutes 40% of myofibrillar protein. Each myosin molecule consists of two identical heavy chains (MyHC, 220 kDa ...each) and two pairs of non-identical myosin light chains. There are four major sarcomeric MyHC isoforms in adult mammalian skeletal muscle, MyHC-2a, -2b, -2x, and "-slow". Each MyHC isoform is encoded by a distinct gene. Individual muscle fibers are characterized according to their MyHC content: type II A fiber (containing MyHC-2a), type II B fiber (containing MyHC-2b), type II X fiber (containing MyHC-2x), and type I fiber. Skeletal muscle fibers can alter their MyHC isoform composition in response to electrical stimuli and mechanical factors such as stretch. However, the molecular mechanisms determining gene-specific activation of these MyHC genes are not well defined. Expression of the four major MyHC isoform genes was reported in rat muscles. Expressions of these four MyHC isoforms in the porcine skeletal muscles were also detected by in situ hybridization and RT-PCR. Expression pattern of a fetal bovine MyHC gene was reported; however, expression pattern MyHC isoform genes in bovine skeletal muscles has not been reported. Cattle are large ruminant mammals, whose physiology is different from that of non-ruminant mammals; therefore, bovine MyHC isoform composition would differ from that of other mammals. Moreover, MyHC isoform composition would also affect the quality of the meat originating from bovine muscles. In this report, we determined the sequences containing 5' non-coding region of bovine MyHC-2a, -2x and -slow isoforms, which differed among the isoforms. Based on these sequences, we developed a procedure to detect the expression of these three MyHC isoforms in bovine muscles by the RT-PCR method with the use of the multiplex PCR technique.
The full amino acid coding sequences of adrenergic receptor genes beta1, beta2, and beta3 (ADRB1, ADRB2, and ADRB3) were determined for Jinhua, Meishan, Duroc and Landrace pigs. Non-synonymous ...substitution of Arg458Pro was found in the porcine ADRB1 gene, resulting in a 469 amino acid sequence. Continuous substitutions of Asn29Asp and Glu30Gln were found in the porcine ADRB2 gene, resulting in a 418 amino acid sequence. Additionally, a Lys30 polymorphism of the ADRB2 gene was found in the Jinhua pigs. There were three non-synonymous substitutions of Asn24Thr, Arg264Gln and Asn398Asp on the porcine ADRB3 gene. A thymine insertion in the ADRB3 gene, resulting in a protein with two fewer amino acids, was found in the Jinhua and Meishan pigs. To assess the effect of ADRB polymorphisms on porcine subcutaneous fat layer thickness, we calculated the genetic frequency of the variants in fatty and lean groups, each consisting of 24 pigs that were crossbreds of Duroc and Jinhua pigs. The effect of the ADRB3 gene polymorphism was not evaluated, because there was insufficient variation on the ADRB3 gene in the examined groups. Although Fisher's exact test showed no significant difference in the frequency of ADRB1 and ADBR2 variants between the two groups, the Arg458 variant of ADRB1 was higher (P=0.11) in the lean group, and pigs in that group had a thinner fat layer than did those with the Pro458 variant. These results imply a possibility of ADRB1 polymorphism as a minor factor in porcine fat layer thickness. The Asp29 variant of ADRB2 was higher in the lean group (P=0.11), and the Glu30 variant was higher in the fatty group (P=0.15), but the Asp29 variant was found only in the Chinese pigs. Thus, the effect of ADRB2 polymorphisms was not clear in this study.
Troponin T (TnT) is one of the myofibrillar proteins that is easily degraded during postmortem aging of pork. In this study, we determined the N-terminal amino acid sequences of TnT degradation ...fragments produced during postmortem aging and by m-calpain hydrolysis. The N-terminal amino acid sequences of TnT fragments produced during postmortem aging were EVHEPEEKPRPKLTAP, EKPRPKLTAPKIPEG, and APKIPEGEKVDF. On the other hand, the N-terminal amino acid sequences of TnT fragments produced by the action of m-calpain were APPPPAEV, EVHEPEEK, and APK. These sequences of degradation fragments could be mapped on fast type TnT isoform 2. The peptide bonds of His37-Glu38 and Thr51-Ala52 in fTnT2 were cleaved during postmortem aging as well as by the calpain hydrolysis; therefore, calpain was concluded to have an important role in TnT degradation during postmortem aging. It was also found that the sourness-suppressing peptide APPPPAEVHEVHEEVH (Okumura et al. Biosci. Biotechnol. Biochem. 2004, 68, 1657-1662) derived from TnT degradation could be produced by the action of calpains on Glu21-Ala22 and His37-Glu38 sites.
PDF
