Bovine Calpastatin Gene Research Articles

한우(Bos taurus coreanae)의 CAST 유전자 내 변이지역 탐색 및 경제형질과의 연관성 분석

Calpastatin (CAST)은 calpain의 억제제 역할을 하는 유전자이며 BTA7에 위치하고 있다. Calpain을 억제하는 CAST 유전자 내 다형성이 육질의 연도에 영향을 미치고 있는 것이 알려져 있다. 또한 여러 연구를 통해 calpain 시스템은 일반 골격근의 성장에 중요한 역할을 하는 것으로 보고되었다. 따라서 본 연구는 한우 집단의 CAST 유전자 내 변이 지역을 탐색하여 경제형질과의 연관성을 분석하기 위하여 실시하였다. CAST 유전자(Accession no. NC_007305) 단일염기변이지역을 탐색 결과 총 5개의 변이(109749T/C, 116151G/A, 109737G/A, 109823T/C, 109926G/A) 지역이 탐색 NCBI에 등록되어 있지 않은 새로운 변이로 본 연구를 통해 탐색되었다. 한우집단 내에서 탐색된 총 5개의 SNP를 대상으로 연관불균형(LD, Linkage disequilibrium) 분석을 수행하였다. 변이들 간의 연관불균형(LD) 정도가 가장 낮은 수준에 있는 2개의 변이지역(109926G/A와 116151G/A, <TEX>$r^2$</TEX>=0.038)을 대상으로 경제형질과의 연관성 분석을 실시한 결과 등심단면적(109926G/A, p<0.05)과 18개월령 체중(116151G/A, p<0.05)에서 유의적인 연관성이 검출 되었다. CAST유전자는 도축 후 숙성과정에서 고기의 연도를 증가시키는데 영향을 미칠 뿐만 아니라 가축의 성장에도 깊은 연관성이 있으며, 본 연구를 통해 CAST 유전자 내 변이는 성장 관련 형질과의 연관성이 유의적인 것으로 확인되었다. A number of studies have shown that the calpain system is important in normal skeletal muscle growth. An increased rate of skeletal muscle growth can result from a decreased rate of muscle protein degradation, and this is associated with a decrease in activity of the calpain system, due principally to a large increase in calpastatin (CAST) activity. The CAST gene, mapped to BTA 7, is considered a candidate gene for beef tenderness and muscle growth. The present study used comparative sequencing of five novel polymorphisms located within exon 20 and 22 of the bovine CAST gene in Hanwoo: exon20- 109737G/A, 109749T/C, 109823T/C, exon22- 116151G/A, intron- 109926G/A. The association of the CAST SNPs with economic traits was studied. The 109926G/A showed a significant effect only on the longissimus muscle area (LMA, p<0.05) in Hanwoo. 109926G/A with the genotype GG had a significantly higher effect on LMA (75.35) than the genotype AA (69.6, p<0.05). Also, the 116151G/A showed a significant effect only on weight at 18 months (W18, p<0.05). 116151G/A with the genotype GG had a significantly higher effect on W18 (428.54) than the genotype AA (408.87, p<0.05).

Association of CAST Gene Polymorphisms with Carcass and Meat Quality Traits in Chinese Commercial Cattle Herds

Calpastatin (CAST), an endogenous inhibitor of the calpains, plays an important role in post-mortem tenderization of meat. The objectives of this study were to investigate single nucleotide polymorphisms (SNPs) in the bovine CAST gene and association with carcass and meat quality traits. A total of 212 cattle from commercial herds were tested in this study including 2 pure introduced breeds, 4 cross populations, and 3 pure Chinese native breeds. Five SNPs were identified at position 2959 (A/G), 2870 (G/A), 3088 (C/T), 3029 (G/A) and 2857 (C/T) in the CAST gene (GenBank Accession No. AF159246). Allele frequencies of SNP2959 and SNP2870 were 0.701 (A) and 0.462 (A), respectively. A general linear model was used to evaluate the associations between the two markers and 7 traits. The results showed that both SNP2959 and SNP2870 were significantly (p<0.01) associated with the Wamer-Bratzler shear force (WBSF), while they had no significant association with the other 6 traits in the whole population. However, in Chinese native pure breeds, only SNP2870 had significant association with WBSF (p<0.05). The simultaneous analysis of two-marker genotype effects indicated animals containing the A/G haplotype (A for SNP2959 and G for SNP2870) tended to have lower shear force than those containing the G/A haplotype, and, especially, animals homozygous for the A/G haplotype had approximately 2 kg lower shear force than those homozygous for the G/A haplotype (p<0.01). These results suggested that both markers may be effective for the marker-assisted selection of meat quality traits in Chinese commercial herds, especially SNP2870 which can be used for Chinese native cattle. I.

Characterization of Bovine Calpastatin Gene in Nelore Cattle Using Polymerase Chain Reaction-Restricted Fragment Length Polymorphisms

Problem statement: In beef cattle production, of meat quality and carcass traits are important. Traditionally beef cattle breeding programs unfortunately are time consuming and also recording of carcass and growth traits need heavy cost, Approach: Marker Assisted Selection (MAS) should be utilized in beef herds, along with economically important phenotypic traits, for genetic progress to made with respect to improving the uniformity and consistency of beef. Blood samples were collected from 41 nelor cattle in Malaysia. Forward and reversed primers amplified a 1552 bp fragment from calpastatin gene. XmnI enzyme was used for restriction analysis of PCR products. Result: Overall, the frequency of alleles A and B in the studied breeds were estimated as 0.42 and 0.58, respectively. In this study we calculated genotype frequency AA, AB and BB 12.2, 58.53 and 29.27% respectively and also observed heterozygosity, expected heterozygosity and average value of heterozygosity were 0.58, 0.49 and 0.48 respectively. Highest frequency of allele was B (0.58) and lowest was A (0.42) This Nelor cattle population was in the Hardy-Weinberg equilibrium. Conclusion: Perhaps, this molecular genetic information helps breeders for designing the proper genetic selection program in the development direction of this breed.

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.

Haplotypic Diversity Within the Ovine Calpastatin (CAST) Gene

Calpastatin (CAST) is a protein inhibitor that acts specifically on calpains and plays a regulatory role in postmortem beef tenderization and muscle proteolysis. Polymorphisms in the bovine CAST gene have been associated with meat tenderness, but little is known about how the ovine CAST gene may affect sheep meat quality traits. In this study, we selected two parts of the ovine CAST gene that have been previously reported to be polymorphic (region 1-part of intron 5 and exon 6, and region 2-part of intron 12), to investigate haplotype diversity across an extended region of the ovine gene. First, we developed a simple and efficient polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) method for genotyping region 2, which allowed the detection of a novel allele as well as the three previously reported alleles. Next, we genotyped both regions 1 and 2 of the ovine CAST gene from a large number of sheep to determine the haplotypes present. Nine different haplotypes were found across this extended region of the ovine CAST gene and four haplotypes were identified that suggested historical recombination events within this gene. Haplotypes are typically more informative than single nucleotide polymorphisms (SNPs) for analyzing associations between genes and complex production traits, such as meat tenderness, but the potential for intragenic recombination within the ovine CAST may make finding associations challenging.

A novel polymorphisms in intron 12 of the bovine calpastatin gene

Calpastatin (CAST) is a specific inhibitor of the ubiquitous calcium-dependent proteases-mu-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.

Four promoters direct expression of the calpastatin gene

Calpastatin is a specific endogenous protein inhibitor of the ubiquitous calcium dependent proteinases μ- and m-calpain. The calpain–calpastatin system is involved in various physiological and pathological processes. In the present study, we determined the bovine calpastatin gene structure and demonstrated that four promoters direct its expression. The gene harbours 35 exons spanning at least 130 kb on genomic DNA. Its structure is similar to that of mouse, pig, and human gene. Transient transfection assays in both C2C12 and COS7 cell lines demonstrated that the putative promoter regions situated 5′ to exon 1xa, 1xb, 1u, and 14t were functional. We also established that the region situated upstream exon 14t is subjected to a tissue specific regulation. The implication of numerous high-scoring cis acting transcriptional motifs which are present in these regions will need to be determined. The existence of four promoters suggests differential expression patterns which must have a physiological significance.

Genetic variants detected by PCR-RFLP in intron 6 of the bovine calpastatin gene.

PCR conditions: A total of 35 cycles were adapted for denaturation at 94 °C/1 min, annealing at 57 °C/1 min, and polymerization at 72 °C/2 min (MJ Research, PT 200, Watertown, MA, USA). A total of 30 ll of mixture consisting of 3 ll of 10X reaction buffer (10 mM Tris, pH 8.3, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2), 10 lM dNTP, 10 pmol of each primer, 50 ng of genomic DNA and 2 units of Taq DNA polymerase (Gibco BRL, Grand Island, NY, USA) was used.

A DNA polymorphism of the bovine calpastatin gene detected by SSCP analysis.

A DNA polymorphism of the bovine calpastatin gene detected by SSCP analysis.

Rapid communication: a polymorphic microsatellite in the promoter region of the bovine calpastatin gene.

Marker Name. CAST. Source and Description of Primers. Primers were designed from a published sequence of the bovine calpastatin promoter region surrounding a cytosine-adenine (CA) repeat 1.4 kb upstream of the transcription start site (Cong et al., 1998), to amplify a product from genomic DNA of approximately 246 bp (primers CSTF5 and CSTR4) that was used for sequencing. A polymorphic 126 to 154 bp amplicon was produced with primers CSTF4 and CSTR4 for microsatellite analysis. Primer Sequences. CSTF4: 5′-GTA AAG CCG CAC AAA ACA CAC CCA GG-3′; CSTF5: 5′-TTC AAC AGC CTC CTG AAA GGC AAT GG-3′; CSTR4: 5′-CCT GGA CCC TCT GGA TGA GGA AGC GG-3′. Method of Detection. The PCR amplification of 50 ng of genomic DNA for cycle sequencing was performed for 35 cycles of 94°C for 30 s, 57°C for 45 s, and 72°C for 45 s, followed by a 5-min final extension at 72°C in an MJ (Watertown, MA) PTC-100 thermocycler. Sequencing was performed as in Geesink et al. (1998). For microsatellite analysis, a total of 25 ng of genomic DNA, 5 pmol of each primer, and .1 U of Taq polymerase were included in an 8-μL reaction containing 1× Taq buffer, 30 μM of dTTP, dGTP, dCTP, and 15 μM dATP, and .1 μCi of [α-32P]ATP. The cycling profile was 1 min at 92°C, with 30 cycles of 30 s at 94°C, 1 min at 55°C, and 1 min at 72°C, followed by a final 5-min extension at 72°C. Description of Polymorphism. The polymorphism was a (CA)n repeat that identified nine alleles in the MARC mapping population (Bishop et al., 1994) with fragment lengths of 126, 132, 136, 142, 146, 148, 150, 152, and

CAMP responsiveness of the bovine calpastatin gene promoter

Previous studies have shown that transcription of the gene encoding bovine calpastatin, an inhibitor of the calcium-activated cysteine protease calpain, is upregulated following activation of cAMP-dependent signaling pathways. In this study, deletion and site-directed mutagenesis experiments were performed to identify cis elements conferring cAMP responsiveness. Heterologous promoter assays demonstrated that all cAMP-responsive cis elements were located within −102 nucleotides (nt) of transcription initiation. Deletion of an element (GTCA) at nt +13 that is identical to half of the palindromic cAMP-responsive element (TGACGTCA) identified in other cAMP-responsive gene promoters had no effect on the response of the calpastatin promoter to dibutyryl-cAMP, although a 67% reduction in basal promoter activity was observed. In contrast, two point mutations in a cis element at nt −76 (GTCA to aTCt) abolished cAMP responsiveness. These results demonstrate that the calpastatin promoter sequence between nt −1653 and +130 contains a single cAMP-responsive element (GTCA) located at nt −76, and suggest a direct molecular pathway by which activation of cAMP signaling could lead to increased calpastatin gene transcription and reduction in calpain-mediated proteolysis.

The Bovine Calpastatin Gene Promoter and a New N-terminal Region of the Protein Are Targets for cAMP-dependent Protein Kinase Activity

To investigate the regulation of calpastatin gene expression, we isolated bovine heart calpastatin cDNAs and 5'-regions of the calpastatin gene. Analysis of 5'-cDNA sequence identified a new translation initiation site that is in frame and 204 nucleotides upstream of the previously designated start site. Conceptual translation from this upstream AUG produces a protein containing 68 additional N-terminal amino acids. This "XL" region contains three potential PKA phosphorylation sites but shares no homology with other regions of calpastatin or with any known protein. Immunoblot studies demonstrated that heart and liver contain a calpastatin protein of 145 kDa on SDS-polyacrylamide gel electrophoresis that comigrates with full-length bacterially expressed calpastatin and calpastatin produced by coupled in vitro transcription-translation from the upstream AUG. An antibody raised against the XL region recognized the 145-kDa band, demonstrating that the upstream AUG is utilized and that the 145-kDa band represents full-length calpastatin in vivo. Transient transfection assays demonstrated that sequence within 272 nucleotides upstream of transcription initiation of the calpastatin gene is sufficient to direct moderate level transcription. Promoter sequences further upstream act to inhibit or stimulate transcriptional activity. Exposure of transfected cells to dibutyryl cAMP resulted in a 7-20-fold increase in promoter activity for constructs containing at least 272 nucleotides of upstream promoter sequence. Deletion analysis indicates that at least one cAMP-responsive element resides within 102 nucleotides of transcription initiation.

Relationship of restriction fragment length polymorphisms (RFLP) at the bovine calpastatin locus to calpastatin activity and meat tenderness.

Restriction fragment length polymorphisms (RFLP) have been identified at the bovine calpastatin locus. The objective of the present study was to determine whether these polymorphisms are related to variations in calpastatin activity or beef tenderness in unrelated animals of mixed breeding. A sample of 83 crossbred steers from sires representing eight different breeds was examined to determine this relationship. A 2.2-kb cDNA coding for domains 2 through 4 plus a 3' untranslated region of bovine skeletal muscle calpastatin was used as a probe for calpastatin RFLP. Polymorphisms were found using the restriction enzymes BamHI and EcoRI. Polymorphic restriction fragments for BamHI were 9.0 and 5.0 kb and for EcoRI were 6.0 and 4.0 kb. Allelic frequencies for BamHI restriction fragments were .53 for the 9.0-kb allele and .47 for the 5.0-kb allele. Allelic frequencies for EcoRI restriction fragments were .43 for the 6.0-kb allele and >57 for the 4.0-kb allele. No polymorphisms were identified using the restriction enzymes BglII, DraI, or PstI. No associations between EcoRI and BamHI RFLP and 24-h calpastatin activity of Warner-Bratzler shear force at 14 d postmortem were detected. Therefore, the polymorphic EcoRI and BamHI restriction sites within the bovine calpastatin locus do not detect DNA sequence differences responsible for variation in calpastatin activity or tenderness of aged beef. Therefore, these polymorphisms cannot be used to predict tenderness of aged beef from unrelated animals of mixed breeding. These results do not exclude the possibility that other DNA sequences in or near the bovine calpastatin gene are responsible for variation in calpastatin activity or meat tenderness. The lack of a relationship between these calpastatin RFLP and meat tenderness must be distinguished from the well-documented relationship between calpastatin activity and meat tenderness. Therefore, further development of calpastatin-based methods for predicting beef tenderness in unrelated animals of mixed breeding should focus on basic factors influencing calpastatin activity at the molecular and cellular level.

Rapid communication: restriction fragment length polymorphisms in the bovine calpastatin gene1

Rapid communication: restriction fragment length polymorphisms in the bovine calpastatin gene1