Genetic characterization of Egyptian buffalo CSN3 gene

Genetic polymorphism k-casein (CSN3) gene was investigated in lactating Egyptian buffalo using nucleotide sequencing. Primer pairs amplified a 453 nucleotide fragment of CSN3 exon IV with an open reading frame of 421 nucleotides encoding 139 amino acids of the mature peptide and 32 nucleotides 3′UTR. Two SNPs (nt-315 C/T and nt-319 C/T) occurred in amplified fragment. These SNPs were reflected at codon 105 (ACC/ATC) and codon 106 (ACC/ACT) which correspond to codon 135 and 136 of the CSN3 mature peptide, respectively. Variation at codon 135 caused a change from ACC (Threonine) versus ATC (Isoleucine) whereas variation at codon 136(Thr/Thr) is a silent mutation. The results show, contrary to previous reports, that Egyptian buffalo has both alleles A (135^ThrACC/136^ThrACC) and B (135^IleATC/136^ThrACT) with allelic frequencies of 0.57 and 0.43, respectively. The Egyptian buffalo genotype frequencies were 0.294 (AA), 0.647 (AB) and 0.058 (BB). The polymorphic site at codon 135 (A[C/T]C) and 136 (AC[C/T]) has C bases in allele A and T bases in allele B, resulting in two haplotypes; 135^Thr(ACC)/136^Thr(ACC) and 135^Ile(ATC)/136^Thr(ACT). The frequency of the former haplotype was 0.57. In this study we investigated the reason why buffalo samples, analyzed by RFLP technique, using HindIII and HinfI used in cattle, were mistakenly identified as BB monomorphic. We suggest the use of restriction enzymes AcuI or Eco57MI to be used in buffalo CSN3 RFLP analysis. Digestion of buffalo CSN3-exon VI fragment (453 bp) with either enzyme will generate two fragments of 339 bp and 114 bp in allele B.     

Molecular genetic characterization of ovine CSN1S2 variants C and D reveal further important variability within CSN1S2

Within this study, the recently identified ovine CSN1S2 variants C and D were characterized at the molecular genetic level. Sequencing of the cDNA and of parts of the DNA identified several sequence differences within CSN1S2*C and D in comparison to CSN1S2*A and B. CSN1S2*C is characterized by two non-synonymous single nucleotide polymorphisms (SNPs) within exon 7 (c.178A>G, c.187G>T) leading to the amino acid substitutions p.Val45Ile and p.Ala48Ser. CSN1S2*D is caused by the SNP c.183G>C, leading to an amino acid replacement at position 46 (p.Arg46Ser). A very common c.527G>A-SNP within exon 15, resulting in the amino acid substitution p.Arg161His and producing the new variant CSN1S2*G, not detectable by isoelectric focusing and previously misidentified as CSN1S2*A, was also identified. On the basis of the identified sequence differences, a new nomenclature is proposed and a possible phylogenetic pathway shown for ovine CSN1S2 variants.     

Characterization of two new alleles at the goat CSN1S2 locus

Two novel alleles at the goat CSN1S2 locus have been identified: CSN1S2(F) and CSN1S2(D). Sequence analyses revealed that the CSN1S2(F) allele is characterized by a G --> A transition at the 13th nucleotide in exon 3 changing the seventh amino acid of the mature protein from Val to Ile. The CSN1S2(D) allele, apparently associated with a decreased synthesis of alpha s2-casein, is characterized by a 106-bp deletion, involving the last 11 bp of the exon 11 and the first 95 bp of the following intron. Methods (PCR-RFLP and PCR) for identification of carriers of these alleles have been developed.     

Characterization of the CHORI-240 BAC clones containing the bovineCSN1S1, CSN2, STATH, CSN1S2 andCSN3 genes

Nineteen BAC clones were identified by hybridization of the bovine genomic BAC library CHORI-240 with mixedCSN1S1- andCSN3-specific probes. Two of the clones were shown to contain the genesCSN1S1, CSN1S2, CSN2, STATH andCSN3, and five were proved to include the genesCSN2, STATH, CSN1S2 andCSN3. These data showed that the BAC contig was established for the whole casein cluster, including all known five genes.     

TSA1 interacts with CSN1/CSN and may be functionally involved in Arabidopsis seedling development in darkness

The COP9 signalosome (CSN) is a multiprotein complex which participates in diverse cellular and developmental processes.CSN1,one of the subunits of CSN,is essential for assembly of the multiprotein complex via PCI (proteasome,COP9 signalosome and initiation factor 3) domain in the C-terminal half of CSN 1.However,the role of the N-terminal domain (NTD) of CSN 1,which is critical for the function of CSN,is not completely understood.Using a yeast two-hybrid (Y2H) screen,we found that the NTD of CSN1 interacts with TSK-associating protein 1 (TSA1),a reported Ca2+-binding protein.The interaction between CSN1 and TSA1 was confirmed by co-immunoprecipitation in Arabidopsis.tsal mutants exhibited a short hypocotyl phenotype in darkness but were similar to wild-type Arabidopsis under white light,which suggested that TSA1 might regulate Arabidopsis hypocotyl development in the dark.Furthermore,the expression of TSA1 was significantly lower in a csnl null mutant (fus6),while CSN1 expression did not change in a tsal mutant with weak TSA1 expression.Together,these findings suggest a functional relationship between TSA1 and CSN1 in seedling development.     

5个山羊品种CSN1S2基因的Alw26I酶切

利用PCR-RFLP技术对西农萨能奶山羊、关中奶山羊、陕南白山羊、安哥拉山羊和波尔山羊5个山羊品种的170个个体的αs 2酪蛋白(CSN1S2)基因进行多态性分析,结果表明：扩增大小为310 bp的片段经限制性内切酶Alw26I酶切后表现多态,且5个山羊品种该基因座位均处于Hardy-Weinberg平衡状态。西农萨能奶山羊、关中奶山羊、陕南白山羊、安哥拉山羊和波尔山羊的基因杂合度/有效等位基因数/Shaanon信息熵/PIC值分别为0.1589/1.1889/0.2955/0.1463, 0.4114/1.6981/0.6017/0.5171,0.1653/1.1980/0.3046/0.1516,0.0646/1.0691/0.1463/0.0625,0.0541/1.0572/ 0.1270/ 0.0526。分析结果显示,关中奶山羊的遗传多样性最丰富,表现为高度多态;其次是西农萨能奶山羊和陕南白山羊,而安哥拉山羊和波尔山羊的遗传变异程度最低。     

Validation of CSN1S1 transcriptional expression,promoter methylation,and prognostic power in breast cancer using independent datasets

Breast cancer ranked second among most frequent cancer in the world playing a significant role in mortality rate. Having prior knowledge on differentially expressed genes in breast cell carcinoma elucidated important indications to understand the molecular mechanism underneath breast carcinogenesis. In this study we have investigated the distinguished CSN1S1 expression in human breast cancer. We have analyzed CSN1S1 mRNA expression between cancer and normal tissues using TCGA datasets. Moreover, analysis including promoter methylation, mutations, prognosis, co-expression, gene ontology, and pathways of CSN1S1 were performed by the TCGA Wanderer, UCSC Xena, cBioPortal, PrognoScan, UALCAN, and Enricher server. We have observed low mRNA expression and high promoter methylation of CSN1S1 in cancer tissues compared to normal tissues. Furthermore, we have also identified low mRNA expression in clinicopathological patients, as well as 9 deleterious mutations with highly co-expressed protein MRC1, and significantly related signaling pathways. We have found a positive correlation between the lower expression of CSN1S1 and patients surviving with breast cancer. Here we have concluded that CSN1S1 acts as a biomarker for the surveillance and prognosis of breast cancer, and also works as a novel therapeutic target at the molecular and pathway levels.     

Improving the reproductive performance of Egyptian buffalo cows by changing the management system

The objective of the present study was to determine the possibility to improve the reproductive performance of buffalo cows through the continuous exposure to bull with grazing and free-stall housing management. Sixty-four Egyptian multiparous buffalo cows raised under two different management systems in two farms were used in this study. The cows in the first farm (management system 1, MS1) were loose--housed in a free-stall yard, grazed for 4 h per day, suckled their calves for 2-3 months and were continuously exposed to a fertile bull. The cows in the second farm (management system 2, MS2 ) were confined in an open-fronted tie-stall shed, not grazed, suckled their calves for only 7 days and were exposed to a fertile bull twice per day (30 min per session). All the cows were fed a diet of green berseem (Trifolium alexandrinum), rice straw and concentrates to meet their maintenance and production requirements. The cows during both the treatments were milked twice per day after weaning. The cows in both groups were between the second and the sixth parity, weighed 450-480 kg and had average daily milk yields of 5.0-6.0 kg. In each farm, cows were visually checked twice daily at 07:00 and 17:00 h for the signs of oestrus and animals proved standing heat were naturally mated. Rectal palpation was used to monitor uterine involution and for pregnancy diagnosis. Blood was sampled twice per week from 7 to 150 days post-partum for serum progesterone assay. The results revealed that post-partum intervals to each of first ovulation, first oestrus, conception and next parturition were significantly (P < 0.05) shorter in MS1 group than in MS2 group. In the meantime, MS1 increased (P < 0.01) the conception and calving rates by 21 and 25%, respectively compared to MS2. Percentages of post-partum cyclic animals and animals exhibiting ovulatory oestrus were greater (P < 0.01) in MS1 group than in MS2 group. However, the percentage of animals cycling before day 60 post-partum was significantly (P < 0.01) lower in MS1 group than in MS2 group (13% versus 28%). By day 120 post-partum, only 63% of the buffaloes were cycling in MS2 group versus 94% in MS1 group. Percentage of silent ovulation was insignificantly higher in MS2 group (34%) than in MS1 group (25%). However, the percentage of false oestrus was higher (P < 0.01) in MS1 group than in MS2 group (16% versus 3%). In addition, percentage of short ovulatory cycles (15-17 days) was greater (P < 0.01) in MS1 group than in MS2 group, whereas percentage of long ovulatory cycle (25-28 days) was higher (P < 0.01) in MS2 group than in MS1 group. It was concluded that continuous exposure of buffalo cows to a fertile bull with grazing management under free-stall housing system enhances resumption of post-partum ovarian activity and improves conception and calving rates.     

A Single Nucleotide Polymorphism and Sequence Analysis of CSN1S1 Gene Promoter Region in Chinese BOS Grunniens (YAK)

The aim of this study was to investigate the polymorphism of the CSN1S1 gene promoter region in 4 Chinese yak breeds, and compare the yak CSN1S1 gene promoter region sequences with other ruminants. A Polymerase Chain Reaction-Single Strand Conformation Polymorphism protocol was developed for rapid genotyping of the yak CSN1S1 gene. One hundred fifty-eight animals from 4 Chinese yak breeds were genotyped at the CSN1S1 locus using the protocol developed. A single nucleotide polymorphism of the CSN1S1 gene promoter region has been identified in all yak breeds investigated. The polymorphism consists of a single nucleotide substitution G→A at position 386 of the CSN1S1 gene promoter region, resulting in two alleles named, respectively, G₃₈₆ and A₃₈₆, based on the nucleotide at position 386. The allele G₃₈₆ was found to be more common in the animals investigated. The corresponding nucleotide sequences in GenBank of yak (having the same nucleotides as allele G₃₈₆ in this study), bovine, water buffalo, sheep, and goat had similarity of 99.68%, 99.35%, 97.42%, 95.14%, and 94.19%, respectively, with the yak allele A_386.     

A meta-analysis on association between CSN3 gene variants and milk yield and composition in cattle

A meta-analysis on the effects of A and B alleles, the most frequent alleles of CSN3 gene, on milk yield and composition traits was conducted by pooling a large dataset consisting of 30 471 genotyped cattle. Four genetic models, comprising dominant (AA + AB vs. BB), recessive (AA vs. AB + BB), additive (AA vs. BB) and co-dominant (AA + BB vs. AB), were employed to analyze data. Standardized mean difference (SMD) was used to measure the size of the effects of A and B alleles of CSN3 on studied traits. Effect sizes of 0.2, 0.5 and 0.8 represent small, medium and large effects, respectively. The results indicate that B allele, in the form of BB genotype, has a significant, but medium effect on lactation yield under dominant (SMD = 0.259, P-value = 0.006) and additive (SMD = 0.279, P-value = 0.035) models. Moreover, a small decrease in the fat percentage occurred in cows having A allele under dominant (SMD = −0.077, P-value = 0.006) and additive (SMD = −0.106, P-value = 0.035) models. Furthermore, CSN3 variants significantly but slightly affect protein percentage under dominant (SMD = −0.146, P-value = 0.000), recessive (SMD = −0.077, P-value = 0.000) and additive (SMD = −0.219, P-value = 0.000) models, showing the negative effect of A allele on this trait. Meta-analysis results reveal that daily milk yield is slightly affected by CSN3 variants under recessive (SMD = 0.056, P-value = 0.033) and additive (SMD = 0.061, P-value = 0.013) genetic models. There is no effect of CSN3 variants on either protein or fat yield. In addition, the effects of CSN3 variants on milk-related traits were not observed under the co-dominant model. Sensitivity and publication bias analyses were carried out to confirm the stability of meta-analyses results.