K. Wicińska

A novel polymorphisms in intron 12 of the bovine calpastatin gene

Calpastatin (CAST) is a specific inhibitor of the ubiquitous calcium-dependent proteases—μ-calpain and m-calpain, found in mammalian tissues. This proteolytic system plays a key role in the tenderization process that occurs during post-mortem storage of meat under refrigerated conditioning. Fragments of the bovine CAST gene including intron 12 were amplified and subjected to SSCP analysis. Four new SNPs were found within intron 12 of the CAST gene: a transition T/C at position 3893+155* A/G at position 3893+163, a transversion T/A at position 3893+223 and a substitution A/G at position 3893+428 (consensus sequence—GenBank AY834771). The genetic variants in the bovine CAST gene can be analyzed with RFLP method and was studied in 375 bulls of six breeds, including Hereford, Aberdeen-angus, Simmental, Charolaise, Limousine and Polish Black-and-White (BW; Fresian) breeds.

Promoter Variant-Dependent mRNA Expression of the MEF2A in Longissimus Dorsi Muscle in Cattle

The myocyte enhancer factor 2A (MEF2A) gene encodes a member of the myocyte enhancer factor 2 (MEF2) protein family that is involved in vertebrate skeletal, cardiac, and smooth muscle development and differentiation during myogenesis. According to recent studies, MEF2 genes might be major regulators of postnatal skeletal muscle growth; thus, they are considered to be important, novel candidates for muscle development and body growth in farm animals. The aim of the present study was to search for polymorphisms in the bovine MEF2A gene and analyze their effect on the MEF2A mRNA expression level in the longissimus dorsi muscle of Polish Holstein-Fresian cattle. In total, 4094 bp of the whole coding sequence and the promoter region of MEF2A were re-sequenced in 30 animals, resulting in the detection of 6 novel variants as well as one previously reported SNP. Three linked mutations in the promoter region (-780T/G, g.-768T/G, and g.-222A/G) and only two genotypes were identified in two Polish breeds (TTA/TTA and TTA/GGG). Three SNPs in the coding region [g.1599G/A (421aa), g.1626G/A (429aa), and g.1641G/A (434aa)] appeared to be silent substitutions and segregated as two intragene haplotypes: GGG and AAA. Expression analysis showed that the mutations in the promoter region are highly associated with the MEF2A mRNA level in the longissimus dorsi muscle of bulls carrying two different genotypes. The higher MEF2A mRNA level was estimated in the muscle of bulls carrying the TTA/TTA (p<0.01) genotype as compared with those with TTA/GGG. The results obtained suggest that the nucleotide sequence mutation in MEF2A might be useful marker for body growth traits in cattle.

Nucleotide sequence and variations of the bovine myocyte enhancer factor 2C (MEF2C) gene promoter in Bos Taurus cattle

Myocyte Enhancer Factor 2 (MEF2) proteins are a small family of transcription factors that play pivotal role in morphogenesis and myogenesis of skeletal, cardiac, and smooth muscle cells. In vertebrates, there are four MEF2 genes, referred to as MEF2A, -B, -C, and -D, that are located on different chromosomes. After birth MEF2A, MEF2B, MEF2D transcriptions are expressed ubiquitously, whereas MEF2C transcripts are restricted to skeletal muscle, brain, and spleen. In this study, on the basis of the sequences of the bovine chromosome 7 genomic contig, available in the GenBank database, sets of PCR primers were designed and to amplify the bovine MEF2C gene promoter region, exon 1 (5′UTR) and part sequence of the intron 1. Seven overlapping fragments of the bovine MEF2C gene were amplified and then sequenced. Altogether, these fragments were composed in the 3,120-bp sequence which was deposited in the GenBank database under accession no. GU211007. The sequence fragment included the putative site of the promoter region and transcription start of the exon 1. The sequence analysis of these fragments in individual animals representing different Bos taurus breeds revealed four variations in promoter region: g.-1606C>T, g.-1336_-1335DelG, g.-818C>T, g.-613_-612DelA and four SNPs within intron 1: g.2711A>G, g. 2913A>G, g.2962G>T and g.3014A>G. No polymorphism was found within sequence of the exon 1 (5′UTR). These polymorphisms were identified for first time using these sequences and were confirmed by RFLP or MSSCP methods.

Identification of the new polymorphisms in the promoter region of the CAST gene in cattle

Calpastatin (CAST) is a specific inhibitor of the ubiquitous calcium-dependent proteases -μ-calpain and m-calpain. This proteolytic system plays a key role in the tenderization process that occurs during postmortem storage of meat under refrigerated conditions. In the present study using comparative sequencing seven novel polymorphisms located within P3 promoter region for exon 1u of the bovine CAST gene: -357 (C/G), -556 (G/T), -557 (A/G), -580 (G/C), -750 (T/C), and two InDel at position -890 (A/-) and (GTT/-) at position -353/-351 were found. This region directs the expression of type III calpastatin mRNA, encoding the prototypical calpastatin. The genotype frequencies and haplotypes distribution were studied in 191 bulls belonging to six cattle breeds. All genotypes were distributed according to the HWE test and two major combined haplotypes were identified. The frequency of the haplotype1 varied from 0.45 in Aberdeen Angus to 0.82 in Simmental.

Postnatal expression patterns and polymorphism analysis of the bovine myocyte enhancer factor 2C (Mef2C) gene.

The aim of this study was to analyze the level of expression of the Mef2C gene in the developing bovine longissimus dorsi (LD) muscle (at 6, 9 and 12months of age) and to evaluate differences in expression among Polish Holstein-Friesian (HO) and Limousine (LM) bulls. Moreover, the expression patterns of Mef2C in different tissues were determined. The results showed that Mef2C mRNA was expressed at a high level in adult skeletal and cardiac muscles. Moreover, Mef2C expression was markedly lower in the semitendinosus (ST) than in the gluteus medius (GM) and LD muscles. A relatively higher Mef2C mRNA and MEF2C protein level was estimated in the muscles of HO bulls at the age of 12months in comparison with its lower expression in LM bulls. Furthermore, we found that the Mef2C promoter variant (GU211004:g.-1606C>T) does not affect the level of mRNA in the LD and ST muscles of 12-month-old HO bulls.

Three New SNPs in Coding and Noncoding Regions of the Bovine CATB Gene

Proteolysis regulation is a key factor in meat manufacturing processes. Cathepsins are a group of acidic lysosomal proteases involved in postmortem muscle proteolysis, which is of utmost importance in meat products. As the aging of meat proceeds, the lysosomal membrane becomes more fragile, allowing cathepsins to leak from the lysosome to the cytosol, and the enzymes are activated at an acidic pH caused by the accumulation of lactic acid in the muscle as a result of postmortem anaerobic glycolysis (O’Halloran et al., 1997). It is suggested that three cysteine proteases, cathepsins B, H, and L, and an aspartic protease, cathepsin D, are involved in the postmortem degradation of myofibrillar proteins (myosin and actin), and cathepsin B is thought to be one of the most important enzymes involved in beef tenderness (Harper, 1999). The CATB gene is mapped to BTA 8 (Sonstegard et al., 2000) and has nine exons spanning approximately 7.3 kb (Mordier et al., 1995). The last exon is sandwiched between Alu-like short-interspersed nuclear elements. Characterization of cDNAs encoding human, mouse, and bovine cathepsin B has revealed that cysteine proteinase is synthesized as a preproenzyme that undergoes a posttranslational processing during its transport to the lysosomal compartment (Mordier et al., 1993). Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. The CATB gene encodes the protease cathepsin B,