Molecular cloning and characterization of buffalo alpha(s1)-casein gene.

Buffaloes in Indian subcontinent play an important role as the producer of milk and milk products. The alpha(s1)-casein constitutes 38% of the total milk proteins. The present study was carried out to characterize the gene in Murrah breed of Riverine buffalo. Buffalo alpha(s1)-casein cDNA was synthesized by RT-PCR, then cloned using pDRIVE-cloning vector and sequenced. The sequencing revealed that the size of alpha(s1)-casein cDNA was of 645 bp with GC content of 45.58%. The alpha(s1)-casein gene coded 214 amino acids precursor with a signal peptide of 15 amino acid residues. The similarity of buffalo alpha(s1)-casein mRNA sequence with that of cattle, goat, sheep, pig, camel, equine and human were estimated as 97.2, 93, 92.3, 57.2, 59.5, 55.9 and 46.6%, respectively. A similar trend was observed when compared amino acid sequences of these species. In the phylogenetic trees, constructed from the data of the alpha(s1)-casein mRNA as well as protein sequences, it has been observed that buffalo, cattle, goat and sheep formed a cluster with a closer relationship between cattle and buffalo followed by goat and sheep.     

Sequence analysis of UTR and coding region of kappa-casein gene of Indian riverine buffalo (Bubalus bubalis).

In this study, complete nucleotide as well as derived amino acid sequence characterization of water buffalo (Bubalus bubalis) kappa-casein gene has been presented. Kappa-casein cDNA clones were identified and isolated from a buffalo lactating mammary gland cDNA library. Sequence analysis of kappa-casein cDNA revealed 850 nucleotides with an open reading frame (ORF) of 573 nucleotides, encoding mature peptide of 169 amino acids. The 5' untranslated region (UTR) comprised 71 nucleotides, while 3' UTR was of 206 nucleotides. A total of 11 nucleotide and seven amino acid changes were observed in, buffalo (Bubalus bubalis) as compared to cattle (Bos taurus), sheep (Ovis aries) and goat (Capra hircus). Among these nucleotide changes, eight were unique in buffalo as they were fully conserved in cattle, sheep and goat. Majority of the nucleotide changes and all the amino acid changes; 14 (Asp-Glu), 19(Asp/Ser-Asn), 96(Ala-Thr), 126(Ala-Val), 128(Ala/Gly-Val), 156(Ala/Pro-Val) and 168(Ala/Glu-Val) were limited to exon IV. Three glycosylation sites, Thr 131, Thr 133 and Thr 142 reported in cattle and goat kappa-casein gene were also conserved in buffalo, however, in sheep Thr 142 was replaced by Ala. Chymosin hydrolysis site, between amino acids Phe 105 and Met 106, important for rennet coagulation process, were found to be conserved across four bovid species. Buffalo kappa-casein with the presence of amino acids Thr 136 and Ala 148 seems to be an intermediate of "A" and "B" variants of cattle. Comparison with other livestock species revealed buffalo kappa-casein sharing maximum nucleotide (95.5%) and amino acid (92.6%) similarity with cattle, whereas with pig it showed least sequence similarity of 76.0% and 53.2%, respectively. Phylogenetic analysis based on both nucleotide and amino acid sequence indicated buffalo kappa-casein grouping with cattle, while sheep and goat forming a separate cluster close to them. The non-ruminant species viz. camel, horse and pig were distantly placed, in separate lineages.     

Molecular characterization and phylogenetic analysis of a yak (<Emphasis Type="Italic">Bos grunniens</Emphasis>) κ-casein cDNA from lactating mammary gland

κ-Casein is one of the major proteins in the milk of mammals. It plays an important role in determining the size and specific function of milk micelles. We have previously identified and characterized a genetic variant of yak κ-casein by evaluating genomic DNA. Here, we isolate and characterize a yak κ-casein cDNA harboring the full-length open reading frame (ORF) from lactating mammary gland. Total RNA was extracted from mammary tissue of lactating female yak, and the κ-casein cDNA were synthesized by RT-PCR technique, then cloned and sequenced. The obtained cDNA of 660-bp contained an ORF sufficient to encode the entire amino acid sequence of κ-casein precursor protein consisting of 190 amino acids with a signal peptide of 21 amino acids. Yak κ-casein has a predicted molecular mass of 19,006.588 Da with a calculated isoelectric point of 7.245. Compared with the corresponding sequences in GenBank of cattle, buffalo, sheep, goat, Arabian camel, horse, and rabbit, yak κ-casein sequence had identity of 64.76–98.78% in cDNA, and identity of 44.79–98.42% and similarity of 53.65–98.42% in deduced amino acids, revealing a high homology with the other livestock species. Based on κ-casein cDNA sequences, the phylogenetic analysis indicated that yak κ-casein had a close relationship with that of cattle. This work might be useful in the genetic engineering researches for yak κ-casein.     

Molecular Cloning,Sequence, and Phylogenetic Analysis of T Helper 1 Cytokines of Pashmina Goats

Cytokines play an important role in regulation of immune responses either in health or disease. In the present study, the cDNAs encoding mature Interleukin (IL)-2, interferon gamma (IFN-γ), and IL-12 p35 and p40 of Pashmina goat were cloned and sequenced. The amino acid sequence was deduced from nucleotide sequence and compared with those available in GeneBank. Mature forms of goat IL-2, IFN-γ, IL-12 p35, and IL-12 p40 composed of 135, 143, 196, and 305 amino acid residues, respectively. Comparison of amino acid sequence of goat IL-2 with sheep, buffalo, cattle, pig, camel, cat, and human sequences showed homology percentages of 100, 97.8, 96.3, 72.4, 72.4, 67.2, and 64.7, respectively. Amino acid sequence of goat IFN-γ showed 98.6, 95.8, 81.1, 81.8, 80.4, and 62.9 percent homology with sheep, bovine, pig, horse, dog, and humans, respectively. Homology ranging from 81.6 to 99% for IL-12 p35 sequences and 85.6 to 100% for IL-12 p40 sequences at amino acid level were observed across these species. Multiple sequence alignment and phylogenetic analysis of goat cytokines revealed close relationship with sheep sequence.     

Detection of Polymorphism and Sequence Characterization of Toll-Like Receptor 7 Gene of Indian Goat Revealing Close Relationship Between Ruminant Species

In this study, approximately 3.4 kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046 amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.     

Detection of polymorphism and sequence characterization of toll-like receptor 7 gene of Indian goat revealing close relationship between ruminant species

In this study, approximately 3.4?kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046?amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.     

In vitro culture of the strobilar stage of Echinococcus granulosus from protoscoleces of human, camel, cattle, sheep and goat origin from Kenya and buffalo origin from India

Protoscoleces from human, camel, cattle, sheep, goat (all from Kenya) and buffalo (from India) hydatid cysts were cultured under identical conditions in vitro using the diphasic culture system of Smyth (1979b). Organisms from all sources grew and segmented in culture. Genital anlagen developed in all cultured worms but further genital differentiation occurred only in cultures of cattle (testes) and camel (testes and genital pore) material. The possible significance of these results is discussed in relation to the general epidemiology of hydatid disease and the potential infectivity of the different strains to man.     

Analysis of conserved microsatellite sequences suggests closer relationship between water buffalo Bubalus bubalis and sheep Ovis aries.

The distribution and evolutionary pattern of the conserved microsatellite repeat sequences (CA)n, (TGG)6, and (GGAT)4 were studied to determine the divergence time and phylogenetic position of the water buffalo, Bubalus bubalis. The mean allelic frequencies of these repeat loci showed a high level of heterozygosity among the euartiodactyls (buffalo, cattle, sheep, and goat). Genetic distances calculated from the allelic frequencies of these microsatellites were used to position Bubalus bubalis in the phylogenetic tree. The tree topology revealed a closer proximity of the Bubalus bubalis to the Ovis aries (sheep) genome than to other domestic species. The estimated time of divergence of the water buffalo genome relative to cattle, goat, sheep, pig, rabbit, and horse was found to be 21, 0.5, 0.7, 94, 20.3, and 408 million years (Myr), respectively. Although water buffaloes share morphological and biochemical similarities with cattle, our study using the microsatellite sequences places the bubaline species in an entirely new phylogenetic position. Our results also suggest that with respect to these repeat loci, the water buffalo genome shares a common ancestry with sheep and goat after the divergence of subfamily Bovinae (Bos taurus) from the family Bovidae.     

Phylogenetical and ontogenetical studies on the molecular weight heterogeneity of bovine serum transferrin

Antitransferrin (Tf) rabbit serum was highly specific: it reacted with Tfs of ruminants, such as European breeds and Zebu breeds of cattle, Bali cattle, banteng, swamp and river types of water buffalo, anoa, goat, sheep, deer, antelope, camel, and giraffe, but did not react with serum of other non-ruminant species, such as pig, wild boar, hippopotamus, horse, rabbit, rat, chicken, etc. Electrophoresis of Tf and immunoglobulin G (IgG) complexes was carried out using sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). Within ruminants, the following species showed two Tf molecules on SDS-PAGE; European and Zebu cattle, Bali cattle, banteng, two types of water buffalo, and two species of anoa. Other ruminants, sheep, goat, deer, antelope, camel, and giraffe, etc., showed only one Tf molecule. The Tf heterogeneity in molecular weight was, thus, restricted to Bos, Bubalus, and Anoa. The molecular weight of Tf of water buffalo was slightly larger than that of cattle on the gel. The peptide pattern from cyanogen bromide cleavage of Tf of the water buffalo differed clearly from that of cattle. Fetal Tf showed only one molecule during development, but a newborn calf has two Tf molecules, (one large and one small) within 18 hr after birth. We suggest, therefore, that the small molecules formed during the last month of gestation. The peptide patterns of adult and fetal Tfs cleaved by cyanogen bromide differed with regard to the two large peptides; fetal Tf, lacking the second-largest peptide, had twice the amount of the largest peptide compared with adult Tf. From these results, we suggest that a change in peptide sequence occurs from the last month of gestation, when the largest peptide is degraded to the second largest. However, a Tf-like protein detected in the liver microsomal fraction has only one molecular size, both in adult and in fetal livers.     

Comparative FISH mapping in river buffalo and sheep chromosomes: assignment of forty autosomal type I loci from sixteen human chromosomes.

Forty autosomal type I loci earlier mapped in goat were comparatively FISH mapped on river buffalo (BBU) and sheep (OAR) chromosomes, noticeably extending the physical map in these two economically important bovids. All loci map on homoeologous chromosomes and chromosome bands, with the exception of COL9A1 mapping on BBU10 (homoeologous to cattle/goat chromosome 9) and OAR9 (homoeologous to cattle/goat chromosome 14). A FISH mapping control with COL9A1 on both cattle and goat chromosomes gave the same results as those obtained in river buffalo and sheep, respectively. Direct G- and R-banding comparisons between Bovinae (cattle and river buffalo) and Caprinae (sheep and goat) chromosomes 9 and 14 confirmed that a simple translocation of a small pericentromeric region occurred between the two chromosomes. Comparisons between physical maps obtained in river buffalo and sheep with those reported in sixteen human chromosomes revealed complex chromosome rearrangements (mainly translocations and inversions) differentiating bovids (Artiodactyls) from humans (Primates).     

青藏高原藏羚羊朊蛋白基因及氨基酸序列分析

羊瘙痒病是一种自然发生的传染性海绵状脑病,它可以在绵羊和山羊羊群之中传播,其疾病的易感性、潜伏期和跨物种传播的能力主要受宿主朊蛋白基因(prion protein gene,PRNP)的影响。研究藏羚羊PRNP有助于明确藏羚羊和羊瘙痒病之间的关系,从而更好地保护藏羚羊。参照GenBank上已经发表的绵羊PRNP序列设计引物,然后对藏羚羊的PRNP序列进行扩增、测序和分析。测序结果显示藏羚羊PRNP序列由771个核苷酸构成,编码256个氨基酸。序列分析结果显示21只藏羚羊PrP(prion protein,PrP)氨基酸序列完全同源,并且与野生型绵羊PrP氨基酸序列完全一致。此外,藏羚羊PrP氨基酸序列与山羊(99.2%)、汤氏瞪羚(99.2%)、印度羚(99.2%)、驯鹿(98%)、马鹿(98%)、家牛(97.7%)和水牛(96.1%)也具有较高的同源性。在对PrP氨基酸序列136、154、171位多态性分析后发现本次收集的21只藏羚羊样本可能和野生型绵羊一样对羊瘙痒病易感。本研究为羊瘙痒病在藏羚羊羊群中可能出现的风险提供了理论依据,提示要加强藏羚羊羊群中羊瘙痒病的监测。     

Experimental infection of the baboon (Papio cynocephalus) with Echinococcus granulosus of camel, cattle, sheep and goat origin from Kenya

In different areas of the world, strains of Echinococcus granulosus have been described which appear to vary in their infectivity, and laboratory primates have been used as indicators of their infectivity to man. This phenomenon was evaluated in Kenya for hydatid material of human, camel, cattle, sheep and goat origin. Viable eggs, produced by experimental infections in dogs with larval material from all the above intermediate hosts, were fed separately to four baboons (Papio cynocephalus) in each case. Baboons were autopsied between 373 and 501 days following infection and the liver, lungs, heart, spleen and kidneys were thoroughly inspected. Hydatid cysts were recovered from two baboons in each of the camel, sheep and goat groups, three baboons in the cattle group and none of the baboons in the human group. Fertile cysts were found in the cattle and goat groups and it is suggested that the baboon could be used as an experimental model for this parasite.     

Identification of goat allotypic determinants and generation of goat-mouse hybridomas secreting goat IgG2

Five allotypic determinants controlled by independent genes have been identified in goat. Of these determinants, four have been detected with alloimmune antisera and one with monoclonal antibodies. The specificities A1, C1 and D1 are lipoproteins; B1 is possibly an alpha 2 macroglobulin and E1 and IgG2. The specificity B1 is not expressed until the age of 3-4 months. The gene controlling the specificity E1 is present at about the same frequency (0.38-0.41) in goat, sheep, cattle and water buffalo. Stable hybridomas secreting goat IgG2 have been obtained by the fusion of goat peripheral lymphocytes with mouse myeloma cells.     

Cloning and characterization of DGAT1 gene of Riverine buffalo.

The present study was carried out to characterize the DGAT1 gene of Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and DGAT1cDNA were synthesized by RT-PCR, then cloned using pDRIVE cloning vector and sequenced. The sequencing revealed that the size of DGAT1 gene was 1470 bp with GC content of 62.30%. The gene encoded for 489 amino acid precursors and that it possessed 32 amino acids signal peptide. The similarity of buffalo DGAT1 mRNA sequence with that of cattle, pig, monkey, human, mice and rat were determined as 98.4, 90.7, 85.4, 85.0, 77.4 and 77.1%, respectively. Phylogenetic tree constructed from the derived DGAT1 protein sequences of 15 different species illustrated a unique branches for mammals, fly, nematode and plants. Among mammals, cattle and buffalo grouped together, whereas swine formed another group in the same branch. Four motifs were predicted in buffalo DGAT1 peptide sequence, one N-linked glycosylation site (246th position), two putative tyrosine phosphorylation site (316 and 261), one putative diacylglycerol binding site (382-392 amino acid position) and a conserved domain MBOAT (membrane bound acyl transferase from 150 to 474 amino acids) with a histidine as an active residue.     

山羊卵泡刺激素α亚基cDNA的分子克隆与序列分析

从新屠宰的雌山羊脑垂体中提取总RNA ,反转录获得cDNA .以此cDNA为模板用PCR法扩增目的片段 ,获得长为 380bp的山羊卵泡刺激素α亚基cDNA片段 .将它克隆至pMD 18 T Verctor.随机挑选 3个阳性重组子进行测序 ,将测序结果与绵羊、牛、猪等多种哺乳动物该基因的核苷酸序列及相应氨基酸序列进行比较 .结果表明 ,山羊卵泡刺激素α亚基基因氨基酸序列与绵羊、水牛的同源性最高 ,达 96 % ,与牛的同源性达 95 % ,与人的同源性较低 ,为 74 % .山羊卵泡刺激素α亚基基因编码区的核苷酸与绵羊的同源性最高 ,达 95 % ,与水牛、牛的同源性达 94 % ,与马和大鼠的同源性较低 ,为 85 % .总体来看 ,在哺乳类动物中FSHα亚基基因同源性还是很高的 .     

A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis

Macpherson C. N. L. and Mcmanus D. P. 1982). A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis. International Journal for Parasitology12: 515–521. The soluble enzyme extracts from protoscoleces obtained from hydatid cysts of human, camel, cattle, sheep and goat origin were compared on the basis of their isoenzyme patterns for GPI and PGM using isoelectric focusing. Consistent GPI and PGM isoenzyme patterns were obtained for larvae of human, camel and sheep material. Cattle material varied occasionally in having an additional cathodic band in some of the GPI patterns. Two distinct isoenzyme patterns were evident in the goat material for both enzymes. The more common goat patterns were similar to those of human, cattle and sheep (Kenya, U.K. and Argentina) material, which were similar to each other. The rare goat patterns were similar to those obtained for camel material. Cyst location in the various intermediate hosts had no effect on the zymograms obtained. Additionally, no alteration in the major banding patterns was observed between the larvae and homologous adults produced by experimental infections. Of 26 naturally infected dogs, 19 produced adult GPI zymograms resembling human/ sheep/goat (common form) experimental infection patterns, three were similar to experimental cattle infections and four had camel/goat (rare form) patterns.     

Sexing river buffalo (Bubalus bubalis L.), sheep (Ovis aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos taurus L.) X- and Y-painting probes

River buffalo, sheep, and goat spermatozoa were cross-hybridized using double color fluorescence in situ hybridization (FISH) with bovine Xcen- and Y-chromosome painting probes, prepared by DOP-PCR of laser-microdissected-catapulted chromosomes, to investigate the possibility of using bovine probes for sexing sperm of other members of the family Bovidae. Before sperm analysis, the probes were hybridized on metaphase chromosomes of each species, as control. Frozen-thawed spermatozoa of cattle, river buffalo, sheep, and goat were decondensed in suspension with 5 mM DTT. Sperm samples obtained from three individuals of each species were investigated, more than 1,000 spermatozoa were scored in each animal. FISH analysis of more than 12,000 sperm revealed high level of sperm with X- or Y-signals in all of the species investigated, indicating FISH efficiency over 99%. Significant interspecific differences were detected in the frequency of aberrant spermatozoa (aneuploid and diploid) between goat (0.393%) and sheep (0.033%) (P < 0.01), goat and cattle (0.096%) (P < 0.5), as well as between river buffalo (0.224%) and sheep (P < 0.5). There was no significant difference between river buffalo and cattle. The present study demonstrated that it is possible to use bovine X-Y painting probes for sexing and analyzing sperm of other species of the family, thus facilitating future studies on the incidence of chromosome abnormalities in sperm as well as on sex predetermination of embryos for the livestock industry. Mol. Reprod. Dev. 67: 108-115, 2004.     

Variability of milk fat globule membrane protein gene between cattle and riverine buffalo.

A study on butyrophilin (BTN) gene was conducted to detect variability at nucleotide level between cattle and buffalo. Hae III PCR-RFLP was carried out in crossbred cattle and it revealed polymorphism at this locus. Three genotypes namely, AA, BB and AB and two alleles were observed with frequencies 0.78, 0.17, 0.04 and 0.87, 0.13, respectively. The sequences of different cattle, buffalo and sheep breeds have been reported in the EMBL gene bank with accession numbers: AY491468 to AY491475. The nucleotides, which have been substituted from allele A to B, were found to be C to G (71st nucleotide), C to T (86th nucleotide), A to T (217th nucleotide), G to A (258th nucleotide), A to C (371st nucleotide) and C to T (377th nucleotide). The nucleotide substitution at 71st, 86th and 377th position of the fragment were expected to be a silent mutation where as nucleotide changes at 217th, 258th and 371st positions were expected to be substituted by lysine with arginine, valine with isoleucine and leucine with proline in allele B. The differences of nucleotides and amino acids between cattle, buffalo and sheep breeds have been revealed and on the basis of nucleotide as well as protein variability the phylogenetic diagram have been developed indicating closeness between cattle and buffalo.