Identification of SNPs,mapping and analysis of allele frequencies in two candidate genes for meat production traits: the porcine myosin heavy chain 2B (MYH4) and the skeletal muscle myosin regulatory light chain 2 (HUMMLC2B)

Myosin is one of the most important skeletal muscle proteins. It is composed of myosin heavy chains and myosin light chains that exist with different isoforms coded by different genes. We studied the porcine myosin heavy chain 2B (MYH4) and the porcine skeletal muscle myosin regulatory light chain 2 (HUMMLC2B) genes. A single nucleotide polymorphism (SNP), identified for each gene, was used for linkage mapping of MYH4 and HUMMLC2B to porcine chromosome (Sscr) 12 and Sscr 3, respectively. The mapping of these two genes was confirmed by using a porcine-rodent radiation hybrid panel, even if for MYH4 the LOD score and the retention fraction were low. Allele frequencies at the two loci were studied in a sample of 307 unrelated pigs belonging to seven different pig breeds. Moreover the distribution of the alleles at these two loci was analysed in groups of pigs with extreme divergent (positive and negative) estimated breeding values (EBV) for four meat production traits that have undergone selection in Italian heavy pigs.     

A polymorphic human myosin heavy chain locus is linked to an anonymous single copy locus (D17S1) at 17p13

Restriction fragment length polymorphisms (RFLPs) have been developed for an adult human skeletal muscle myosin heavy chain gene which was previously mapped to the short arm of human chromosome 17. Using RFLP analysis of DNA from 140 individuals, we have found tight linkage (LOD score of 6.9) between this myosin heavy chain gene and the anonymous DNA probe, D17S1.     

Localization of the villin gene on human chromosome 2q35-q36 and on mouse chromosome 1

Summary A partial cDNA clone coding for the 110 carboxyterminal amino acids of human villin was used for mapping the human villin gene. In situ hybridization experiments on human chromosomes with tritiated probe allowed the regional localization of the villin locus to chromosome 2 at q35-36. Data obtained from restriction fragment length polymorphism analysis of two mouse species demonstrated the assignment of the villin gene to mouse chromosome 1 by assessment of linkage with the fast skeletal isoform of the myosin light-chain gene. These villin gene localizations add a fourth locus to the conserved gene cluster encoding the fast skeletal muscle isoform of the myosin light chain, isocitrate dehydrogenase, and the crystallins and confirm the partial homology of the human chromosome 2 long arm and mouse chromosome 1.     

Mutations in the slow skeletal muscle fiber myosin heavy chain gene (MYH7) cause laing early-onset distal myopathy (MPD1)

We previously linked Laing-type early-onset autosomal dominant distal myopathy (MPD1) to a 22-cM region of chromosome 14. One candidate gene in the region, MYH7, which is mutated in cardiomyopathy and myosin storage myopathy, codes for the myosin heavy chain of type I skeletal muscle fibers and cardiac ventricles. We have identified five novel heterozygous mutations--Arg1500Pro, Lys1617del, Ala1663Pro, Leu1706Pro, and Lys1729del in exons 32, 34, 35, and 36 of MYH7--in six families with early-onset distal myopathy. All five mutations are predicted, by in silico analysis, to locally disrupt the ability of the myosin tail to form the coiled coil, which is its normal structure. These findings demonstrate that heterozygous mutations toward the 3' end of MYH7 cause Laing-type early-onset distal myopathy. MYH7 is the fourth distal-myopathy gene to have been identified.     

Human cardiac myosin heavy chain genes and their linkage in the genome.

Human myosin heavy chains are encoded by a multigene family consisting of at least 10 members. A gene-specific oligonucleotide has been used to isolate the human beta myosin heavy chain gene from a group of twelve nonoverlapping genomic clones. We have shown that this gene (which is expressed in both cardiac and skeletal muscle) is located 3.6kb upstream of the alpha cardiac myosin gene. We find that DNA sequences located upstream of rat and human alpha cardiac myosin heavy chain genes are very homologous over a 300bp region. Analogous regions of two other myosin genes expressed in different muscles (cardiac and skeletal) show no such homology to each other. While a human skeletal muscle myosin heavy chain gene cluster is located on chromosome 17, we show that the beta and alpha human cardiac myosin heavy chain genes are located on chromosome 14.     

Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter.     

Localization of human cardiac beta-myosin heavy chain gene (MYH7) to chromosome 14q12 by in situ hybridization

The genes coding for each human cardiac myosin heavy chain (alpha-MHC and beta-MHC, MYH6 and MYH7, respectively) are tightly linked and the alpha-MHC gene has been assigned to chromosome 14. In order to provide a more precise regional localization, in situ hybridization experiments were carried out using a 3H-labeled probe derived from a beta-MHC genomic clone. The results demonstrated that the human cardiac MHC genes are located within the q12 band of chromosome 14.     

肌球蛋白重链基因在人类遗传性疾病中的研究进展

肌球蛋白超家族通过水解ATP,将化学能转化为机械能,在细胞迁移、肌肉收缩等多种生理活动中发挥重要的作用。其中,肌球蛋白Ⅱ类分子是肌细胞和非肌细胞中肌丝的重要组成成分。一个完整的肌球蛋白Ⅱ类分子是由2条肌球蛋白重链(myosin heavy chain, MyHC)和2对不同的轻链组成的六聚体。在人体中,存在多种MyHC亚型,分别由不同的MYH基因家族成员编码。迄今为止,人们已经发现MYH基因家族中多个成员的不同突变与人类遗传性疾病相关。其中,MYH2突变可以导致一类以眼肌麻痹为主要特征的骨骼肌疾病;MYH3和MYH8突变可以引起远端关节挛缩综合征;MYH7突变即可以引起骨骼肌疾病包括肌球蛋白沉积性肌病和Laing远端肌病,也与肥厚性心肌病的发生密切相关;MYH9突变可以导致一类以巨大血小板、血小板减少和中性粒细胞包涵体为特征的MYH9相关性疾病。本文简要介绍MYH基因的表达特点,着重阐述MYH基因与人类遗传性疾病之间的相关性及研究进展。     

Multiple restriction fragment length polymorphisms in the porcine calcium release channel gene (CRC): assignment to the halothane (HAL) linkage group

Two cDNA probes for the porcine calcium release channel gene (CRC) were used in restriction fragment length polymorphism (RFLP) analysis in an attempt to develop genetic markers linked to the malignant hyperthermia (stress susceptibility) gene (HAL). Three TaqI RFLPs, denoted CRC1-CRC3, each composed of two alleles, were detected. RFLPs were also detected with MspI and PvuII, but the MspI RFLP correlated completely with CRC3 in this material and the PvuII RFLP could not be scored reliably due to a minute size difference between the two allelic fragments. The autosomal codominant inheritance of these RFLP loci was confirmed by family analyses. Significant evidence for genetic linkage between the CRC1/CRC3 loci and the A1BG locus in the HAL linkage group confirmed a previous assignment of the CRC gene to chromosome 6 in the pig.     

Mapping of porcine genetic markers generated by representational difference analysis

Representational difference analysis (RDA) was performed using pig genomic DNA from a Landrace non-selected control population and a Landrace population selected for increased loin muscle area (LMA) for five generations. Pigs used for the analysis differed phenotypically for various carcass traits and were divergent in genotype at the skeletal muscle ryanodine receptor 1 locus. Two RDA experiments were performed using BamHI and BglII. Fourteen BamHI and 37 BglII difference products were cloned and sequenced. Oligonucleotide primers were designed to amplify RDA difference products and sequence-tagged sites (STS) were developed for 16 RDA fragments (two BamHI and 14 BglII). These 16 STS were mapped using the INRA-Minnesota porcine Radiation Hybrid panel. Polymorphisms identified in nine of the STS were used to place these markers on the PiGMaP genetic linkage map. Sequence-tagged sites were localized to 11 different chromosomes including three markers on chromosome 11 and four markers on chromosome 14. Development of RDA markers increases the resolution of the pig genome maps and markers located within putative quantitative trait locus (QTL) regions can be used to refine QTL positions.     

Localization of a DNA segment encompassing four tRNA genes to human chromosome 14q11 and its use as an anchor locus for linkage analysis

The chromosomal location of an 8.2-kb genomic fragment encompassing a cluster of four human tRNA genes has been determined by three complementary methods including Southern analysis of human/rodent somatic cell hybrids, in situ hybridization, and genetic linkage analysis. This tRNA cluster (TRP1, TRP2, and TRL1) is located near the T-cell receptor alpha (TCRA) locus at 14q11, and several RFLPs were detected at this site. These RFLPs and those at the TCRA and MYH7 (cardiac beta-MHC gene) loci have been used to type all informative members of the CEPH pedigrees. This has permitted ordering of these three gene loci and two anonymous probes (D14S26 and D14S25) in a 20-cM interval just below the centromere of chromosome 14. Based upon the chromosomal location and the polymorphisms at this site, one or more members of this gene cluster could serve as a useful anchor locus on chromosome 14.     

Localization of a locus for Charcot-Marie-Tooth neuropathy type Ia (CMT1A) to chromosome 17

Phenotypic data for 71 genetic markers for members of five Caucasian kindreds were tested for linkage with the autosomal dominant mutations causing Charcot-Marie-Tooth (hereditary motor sensory) neuropathy type I, characterized by markedly reduced nerve conduction velocities. Lod score analysis gave no evidence of linkage to the closely linked chromosome 1 loci SPTA1-FY-F5-AT3 and APOA2. In contrast, these mutations were found to map closely (zeta = 10.828, theta = 0.0) to D17S58, an anonymous segment of DNA from 17p11.2-p11.1, and thus define the CMT1A locus. Segregation information data for an inferred recombinant offspring indicated that the CMT1A locus is probably proximal to MYH2, the locus encoding adult skeletal muscle myosin heavy polypeptide 2, which maps to 17p13. Analysis of the lod scores on a per kindred basis gave no evidence of genetic heterogeneity.     

Mapping muscle protein genes by in situ hybridization using biotin-labeled probes.

The genes coding for the myosin heavy chain isoforms (unc-54, myo-1, myo-2 and myo-3) and the actins (act-1,2,3 and act-4) have been mapped on the embryonic metaphase chromosomes of Caenorhabditis elegans by in situ hybridization. The genes were cloned in a cosmid vector and the entire cosmid was nick translated to incorporate biotin-labeled dUTP. This produced a probe DNA complementary to a 35-45 kb length of chromosomal DNA. The hybridization signal from the cosmid probe, detected by immunofluorescence, could be easily seen by eye. The clear signals and the specific hybridization of the cosmid probes provided a faster means of mapping these single copy genes than small probes cloned in plasmid or lambda vectors. The myosin heavy chain genes are not clustered. Only unc-54 and myo-1 mapped to the same chromosome; the unc-54 locus is at the extreme right end of linkage group I and myo-1 mapped 40-50% from the left end of linkage group I. Myo-2 mapped to the X, 52-75% from the left end. The myo-3 gene mapped to the middle of linkage group V near the cluster of three actin genes (act-1,2,3). The fourth actin gene, act-4 mapped to 20-35% from the left end of X.     

The porcine proteasome subunit A4 (PSMA4) gene: isolation of a partial cDNA, linkage and physical mapping

A partial cDNA clone encoding the porcine proteasome subunit A4 ( PSMA4 or proteasome subunit C9) has been isolated from a porcine muscle cDNA library and sequenced. A biallelic Taq I RFLP was identified in Large White, Landrace and Duroc breeds. Moreover, the 3'-untranslated region of the gene showed a triallelic SSCP. By linkage analysis the PSMA4 locus was assigned to pig chromosome 7 and by radioactive in situ hybridization this locus was mapped to the region 7q13–q14.     

Porcine malignant hyperthermia carrier detection and chromosomal assignment using a linked probe

In pigs, the gene for glucosephosphate isomerase (GPI) is linked to the halothane (HAL) gene which is responsible for malignant hyperthermia (MH). A single copy DNA probe, designated GPI8R, has been isolated from a pig genomic library using a porcine GPI cDNA probe. This probe detects, as was the case for the cDNA probe, a five allele polymorphism in SacI and PvuII digested pig DNA. Family studies show that this polymorphism is linked to the HAL locus and hence can be used in carrier detection. In situ hybridization with GPI8R assigned the GPI locus to bands p12-q22 of chromosome 6. We conclude that the HAL linkage group resides on chromosome 6.     

The genes coding for the muscle contractile proteins, myosin heavy chain, myosin light chain 2, and skeletal muscle actin are located on three different mouse chromosomes.

The chromosomal distribution of murine genes expressed during differentiation of skeletal muscle cells was determined by Southern blot analysis of DNA from mouse-Chinese hamster hybrid cell lines containing incomplete subsets of mouse chromosomes. All detectable myosin heavy chain genes are located on chromosome 11. The gene for the myosin light chain 2 is located on chromosome 7. The skeletal muscle alpha-actin gene and several other actin genes, or pseudogenes, are located on chromosome 3. Additional actin DNA sequences are distributed on other mouse chromosomes.     

A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle

A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development.     

Two different forms of beta myosin heavy chain are expressed in human striated muscle

Summary We have found evidence for two beta-like myosin heavy chains in humans, one cardiac and one skeletal. The cDNA sequences of the cardiac beta myosin heavy chain cDNA clone pHMC3 and the skeletal beta-like myosin heavy chain cDNA clone pSMHCZ, were compared to each other. It was found that the 3 untranslated regions as well as 482 nucleotides specifying the carboxyl coding region, were 100% homologous. Further examination revealed that the skeletal clone pSMHCZ diverges from the human cardiac beta myosin heavy chain cDNA clone pHMC3 at the 5 end. We present evidence in this report which indicates that the cardiac beta myosin heavy chain mRNA is expressed in skeletal muscle tissues. The human cardiac beta myosin heavy chain cDNA clone, pHMC3, which codes for a portion of the light meromyosin section of the myosin heavy chain, was used as a probe for S1 nuclease mapping studies with RNA derived from cardiac tissue, smooth muscle and skeletal muscle tissues consisting of fast-twitch, slow-twitch and mixed fast- and slow-twitch muscle fibres. Two probes were used to examine the expression of the mRNA. One probe (406 nucleotides) constitutes the 3 untranslated region and a portion of the coding region of the beta cardiac myosin heavy chain cDNA clone, which is 100% homologous to pSMHCZ, the skeletal cDNA clone. The other constitutes the majority of the coding region (1017 nucleotides) of the cardiac clone pHMC3 in which the first 216 nucleotides from the labelled end are 100% homologous to the skeletal clone pSMHCZ. In the soleus muscle, which is rich in slow-twitch type I muscle fibres, the expression of the cardiac beta myosin heavy chain mRNA was very prominent. In gastrocnemius muscle, a mixed fibre muscle, the expression of this mRNA was detected to a lesser degree than that for the soleus muscle. In vastus lateralis and vastus medialis, which consist of predominantly type II, fast-twitch fibres, there were trace amounts of the cardiac beta myosin heavy chain mRNA. When expression of this mRNA was tested in smooth muscle tissue none could be detected.     

Assignment of the porcine major histocompatibility complex to chromosome 7 by in situ hybridization

The major histocompatibility complex (SLA) of the domestic pig (Sus scrofa) was regionally mapped to 7p12----q12 by in situ hybridization with an SLA class I-specific recombinant DNA probe. This localization contradicts linkage data suggesting a possible assignment of the SLA locus to porcine chromosome 15.     

Genetic map of nine polymorphic loci comprising a single linkage group on rat chromosome 10: evidence for linkage conservation with human chromosome 17 and mouse chromosome 11.

Seven genes and two anonymous markers were mapped to a single linkage group on rat chromosome 10 using progeny of an F2 intercross of Fischer (F344/N) and Lewis (LEW/N) inbred rats. Two genes, the neu oncogene or cellular homologue of the viral oncogene erbb2 (ERBB2) and growth hormone (GH) were mapped by Southern blot analysis of restriction fragment length polymorphisms. Five genes, embryonic skeletal myosin heavy chain (MYH3), androgen binding protein/sex hormone binding globulin (SHBG), asialoglycoprotein receptor (hepatic lectin)-1 (ASGR1), ATP citrate lysase (CLATP), and pancreatic polypeptide (PPY), and two anonymous markers, F16F2 and F10F1, were mapped using PCR amplification techniques. The PCR-typable polymorphic markers for the five genes were also highly polymorphic in 10 other inbred rat strains (SHR/N, WKY/N, MNR/N, MR/N, LOU/MN, BN/SsN, BUF/N, WBB1/N, WBB2/N, and ACI/N). These markers should be useful in genetic analysis of traits described in inbred rat strains, as well as in genetic monitoring of such strains. The loci in this linkage group covered 50 cM of rat chromosome 10 with the following order: MYH3, SHBG/ASGR1 (no recombinants detected), F16F2, ERBB2, CLATP, PPY, GH, and F10F1. Comparative gene mapping analysis indicated that this region of rat chromosome 10 exhibits linkage conservation with regions of human chromosome 17 and mouse chromosome 11.