Calpains in Cyanobacteria and the Origin of Calpains

Calpains are cysteine proteases involved in many cellular processes. They are an ancient and large superfamily of enzymes responsible for the cleavage and irreversible modification of a large variety of substrates. They have been intensively studied in humans and other mammals, but information about calpains in bacteria is scarce. Calpains have not been found among Archaea to date. In this study, we have investigated the presence of calpains in selected cyanobacterial species using in silico analyses. We show that calpains defined by possessing CysPC core domain are present in cyanobacterial genera Anabaena, Aphanizomenon, Calothrix, Chamaesiphon, Fischerella, Microcystis, Scytonema and Trichormus. Based on in silico protein interaction analysis, we have predicted putative interaction partners for identified cyanobacterial calpains. The phylogenetic analysis including cyanobacterial, other bacterial and eukaryotic calpains divided bacterial and eukaryotic calpains into two separate monophyletic clusters. We propose two possible evolutionary scenarios to explain this tree topology: 1) the eukaryotic ancestor or an archaeal ancestor of eukaryotes obtained calpain gene from an unknown bacterial donor, or alternatively 2) calpain gene had been already present in the last common universal ancestor and subsequently lost by the ancestor of Archaea, but retained by the ancestor of Bacteria and by the ancestor of Eukarya. Both scenarios would require multiple independent losses of calpain genes in various bacteria and eukaryotes.

Association of SNP g.232 G>T Calpain Gene with Growth and Live Meat Quality Prediction using Ultrasound Images in Bali Cattle

Bali cattle (<em>Bos javanicus</em>) are native Indonesian cattle, domesticated from banteng (<em>Bibos banteng</em>). Genes that have an important role in meat quality are calcium-activated neutral protease genes, known as calpains (CAPN). The objective of this study was to evaluate the polymophisms of calpain gene SNP g.232 G&gt;T by PCR-RFLP technique and its influence on growth trait and meat quality of Bali cattle detected by ultrasound imaging of <em>longissimus dorsi</em> thickness (LDT), back fat thickness (BFT), marbling score (MS), and intramuscular fat percentage (PIMF). The polymorphisms of CAPN1 gene were analyzed by PCR-RFLP using BglII restriction enzyme (n=52 cattle). The ultrasound images of longissimus dorsi muscle were carried out transversally and longitudinal between 12^th -13^ththoracic vertebrae then analyzed by Image-J NIH software. Result showed that SNP g.232 G&gt;T of CAPN1 gene was polymorphic in Bali cattle. SNP g.232 G&gt;T of CAPN1 gene in Bali cattle has higher diversity which was showed of 0.48 heterozygosity value and was in Hardy-Weinberg equilibrium. The polymorphisms of SNP g.232 G&gt;T was associated significantly (P&lt;0.05) with bodyweight at 730 days, marbling score (MS), and intramuscular fat percentage (PIMF). It suggests that the CAPN1 gene in Bali cattle is a candidate for Marker Assisted Selection (MAS), which influences body weight at 730 days, marbling score, and percentage of intramuscular fat.

Analysis of polymorphism of CAPN1 and GH genes in cattle of Kazakh White-Headed breed in association with the peculiarities of beef productivity

The intensive development of genetics at the molecular level has now opened up the possibility for breeders to identify and study genes that are directly or indirectly related to desirable economically useful traits. The purpose of the research was to identify desirable genotypes in the breeding of Kazakh White-Headed cattle based on the genes-markers of meat productivity of calpain (CAPN1) and somatotropin (GH) for use in breeding to increase meat productivity and beef quality. Experimental researches have been carried out on steers of the Kazakh White-Headed breed on the basis of the APC collective farm “Gigant” in the Blagodarnensky district in the Stavropol Territory. Analysis of growth and development indicators of calves has shown that the value of live weight and average daily gains was higher in calves carrying the C allele at calpain gene locus and V allele at somatotropin gene locus. Dominant alleles C and V in the homozygous state characterize the desired genotype of calves. During weaning the studied indicators were higher with a diff erence on average up to 13,0 % in young animals of homozygous genotypes (CC and VV). The largest number of muscle fi bers has been observed in steers with the desired CC and VV genotypes. They outperformed their herdmates with GG genotype by 36,82 pcs/мм² or 19,7 % in calpain gene group (CAPN1) by 55,7 pcs/мм² or 31,4 % in somatotropin gene group (GH), respectively. The diameter of muscle fibers in carcasses with the desired genotype was less by 4,23 microns or 11,9 %, and by 6,82 microns or 16,8 %. The meat of steers with homozygous CC and VV alleles according to the studied genes differed from the meat of animals with GG and LL genotypes by 12,6 % in calpain gene (CAPN1) and 15,1 % in somatotropin gene (GH). The connective tissue content was higher in animals with the undesirable type of G and L alleles than in animals of the desired genotype, by 2,13 and 3,34 abs. percent’s. The area of the eye of loin in animals of desired VV genotype of somatotropin cluster (GH) was higher than in animals of CC genotype of calpain cluster (CAPN1) by 4,84 cм² or 10,1 %.

Single Nucleotide Polymorphism (SNP) 316 on Calpain Gene in Aceh Cattle

Genetic polymorphisms of μ-calpain, a calcium-dependent protease specifically expressed in muscle and related to meat tenderness, have been investigated in cattle worldwide, but not well documented in Indonesian beef cattle. This study was done to evaluate the frequency of SNP 316 in the μ-calpain gene in Aceh cattle, a local Indonesian beef cattle plays important role in the fulfillment of red meat for peoples in Aceh and its neighboring areas. For this purpose, genomic DNA was isolated from 29 sirloin meats samples collected from cull, female aceh cattle slaughtered at the Slaughter House of Banda Aceh. The cattle were purposively selected based on physical characteristics and hair colors referred to Indonesian law. For the SNP genotyping, PCR-RFLP methods were set up. The results showed all three genotypes namely CC, CG, and GG were found in Aceh cattle. The allele and genotype frequencies of SNP CAPN316 in the CAPN gene were: C – 0.14 and G – 0.86; CC – 3.4%, CG – 20.7% and GG – 75.9%. The observed and expected frequencies of CAPN SNP 316 in the cattle population examined were GG 22.0 and 21.6, CG 6.0 and 6.9, and CC 1.0 and 0.6.

CHARACTERISTICS OF UKRAINIAN GREY CATTLE BY DNA-MARKERS

Today conservation of biodiversity is one of the most important priorities in the world. Polymorphism of farm animals is a key to successful breeding; it provides animal adaptation to environmental changes. Impoverishment of genetic resources of farm animals can lead to various negative consequences, such as significant decrease of the effectiveness of selection, existing breeds will not be able to successfully resist to infectious agents evolving constantly; valuable material for the study and analysis of breed origin will be lost. Local breeds created by national selection are valuable genetic resources. Lacking high performance in most cases, they are usually characterized by high resistance to various diseases. Ukrainian Grey cattle, as a representative of the local native breeds, are an interesting object of population research in respect not only of adaptive characteristics, but also genetic mechanisms providing phenotypic expression of certain features of productivity. Recent advances in molecular genetics have made it possible to identify genes associated with qualitative and quantitative characteristics of cattle. The most informative one in this regard is the DNA marker systems based on structural analysis of polymorphisms of genes involved in formation and operation of economically useful traits. The most common potential DNA markers of characteristics of cattle productivity include genes of growth hormone (bGH), beta-lactoglobulin (βLG), thyroglobulin (TG5), calpain (CAPN). BGH gene is an important regulator of somatic growth of animals and has lactating and fat-mobilizing effects. CSN3 gene is linked to milk protein ability and technological properties of milk. Different allelic variants of βLG gene are associated with high content casein and whey proteins in milk, fat percentage and positive impact on milk production. Milk products and falsification detection of milk are controlled by this gene. Its role has been proved in antimicrobial activity to pathogens of mastitis. TG5 gene is a precursor of tyreoid hormones such as triiodothyronine and tetraiodothyronine participating in formation of fat cells and the formation of meat marbling. CAPN gene is involved in proteolysis during ripening of meat and leads to higher tenderness of meat. The aim was to investigate the allelic polymorphism of genes of growth hormone, beta-lactoglobulin, thyroglobulin and calpain in Ukrainian Grey cattle. Blood samples (n = 136) from Ukrainian Grey cattle at "Markeyevo" breeding farm (Kherson region) were studied. Molecular genetic studies were conducted at Laboratory of Genetics of Institute of Animal Breeding and Genetics nd. a. M.V. Zubets of NAAS. DNA isolation from whole blood was performed using standard commercial kit "DNA-Sorb-B" (produced by AmpliSens, Central Research Institute of Epidemiology, Russia). Polymorphisms of genes of GH, βLG5, TG and CAPN1530 was investigated by PCR-RFLP. Electrophoretic separation of restriction fragments of DNA was performed in 1.5% and 2% agarose gels in tris-borate electrophoresis buffer. The results of DNA testing of beta-lactoglobulin locus for A- and B-allele variants in animals of Ukrainian Grey breed found that most of the cows were carriers of homozygous BB genotype of βLG gene. It was determined at every second investigated animals. Homozygous AA genotype was found only at two cows or it is 4%. AB genotype was represented at 45% of animals. Thus, the study found a significant prevalence of frequencies B allele over A allele (0.736 and 0.264, respectively), which leads to overwhelming homozygotisation of one of these alleles. B allele in beta-lactoglobulin gene is associated with high content of casein protein in milk and a high percentage of fat. The values of expected and observed heterozygosity at this gene were not significantly different. The study of gene polymorphism of thyroglobulin found that at the population of the represented breed, the most often seen heterozygous genotype was CT, carriers of which were 57% of the animals. In homozygous state C allele was in 31% of the animals, and T allele – only in 11%. Regarding the level of heterozygosity, we received that actual heterozygosity exceeded theoretically expected one. However, difference is statistically insignificant. Consequently, for thyroglobulin gene Ukrainian Grey cattle belong to breeds that carry the highest frequency of desired C allele for marbling of meat. The analysis of gene polymorphism of bGH indicates the absence of the animals with genotype VV in the investigated sampling, low percentage of heterozygotes (3%) and a significant portion of homozygotes for L allele (98%). Significant differences on levels of actual and expected heterozygosity for somatotropin gene we haven’t found. One of the markers of quality characteristics of meat productivity of cattle is CAPN gene. In general, the animals of Ukrainian Grey breed are characterized by the absence of polymorphism for calpain gene. All the studied animals were carriers of homozygous genotype for preferred G allele (1,0). This feature of the genetic structure of the studied population by calpain gene, animals of which are reproduced in a small array, shows breed-specific high genetic potential by quality characteristic of meat productivity, namely, the tenderness of meat. It was revealed that the population of Ukrainian Grey breed was characterized by low level of polymorphism for genes which cause milk production and meat quality indicators. Thus, homozygous genotype BB dominated for beta-lactoglobulin gene; homozygous LL genotype (98%) was also significantly prevalent and VV genotype was not detected for growth hormone gene; only genotype GG was generally found for calpain gene. And heterozygous CT genotype (57%) dominated only for TG5 locus of thyroglobulin gene.