Polymorphisms in MHC-DRA and -DRB alleles of water buffalo (Bubalus bubalis) reveal different features from cattle DR alleles

Seventy-five individuals of Bubalus bubalis belonging to four different breeds, three of river buffalo and one of swamp buffalo, were studied for polymorphism in MHC DRB (Bubu-DRB) and DRA (Bubu-DRA) loci. Eight alleles of Bubu-DRB were found, and all alleles in the swamp type were shared with the three river breeds. All alleles sampled from the breed of European origin (Mediterranean) were present in breeds sampled in Brazil, thus variability of this locus may have been preserved to a great extent in the more recently founded Brazilian population. Bubu-DRB alleles contained higher proportions of synonymous vs. non-synonymous substitutions in the non-peptide-binding sites (PBS) region, in contrast to the pattern of variation found in BoLA-DRB3, the orthologous locus in cattle. This indicated that either the first domain exon (exon 2) of Bubu-DRB has not undergone as much recombination and/or gene conversion as in cattle alleles, or Bubu-DRB may be more ancient than BoLA-DRB3 alleles. Phylogenetic analysis of DRB alleles from Bubalus, Syncerus c. caffer, the Cape buffalo, and domestic cattle demonstrated transspecies polymorphism. Water buffalo contained two alleles of DRA that differed from each other in two amino acid positions, including one in the PBS (alpha22) that was also shared with Anoa depressicornis, the anoa. Discovery of variation in DRA was surprising as the first domain of DRA is a highly conserved polypeptide in mammals in general and especially in ruminants, where no other substitution in PBS was seen.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

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.     

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Isolation and sequence characterization of mammary derived growth inhibitor gene of riverine buffalo (Bubalus bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5' UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3' UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI/fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Cloning and characterization of novel cathelicidin cDNA sequence of Bubalus bubalis homologous to Bos taurus cathelicidin-4.

Cathelicidin synthesized by bone marrow cells plays an important role in neutralizing invading pathogens. In the present study, the myeloid cathelicidin cDNA from Bubalus bubalis has been cloned and characterized. RNA from bone marrow of buffalo ribs was extracted, reverse transcribed and amplified using specific pair of primer designed from published cathelicidin-4 cDNA sequences of Bos taurus popularly known as indolicidin. An expected amplified product of 517 bp was obtained, which was cloned and sequenced. Comparison of buffalo cathelicidin and indolicidin sequences reveal that the open reading frames (ORF) of both these two congeners consist of 435 nucleotides with 28 divergent nucleotides and the translated proteins of 144 amino acid residues. Fourteen amino acid residues were found to be dissimilar between these two congeners. The molecular mass of buffalo cathelicidin calculated from the deduced amino acid sequence was 16.23 kDa, which is in close proximity of indolicidin. The sequence comparison with known B. taurus cathelicidin congeners again show 70.8-92.9% identity at nucleotides level and 65-88.3% identity at amino acids level. The maximum similarity of buffalo cathelicidin both at nucleotides level (92.9%) and protein level (88.3%) was found to be with indolicidin. Phylogenetic tree analysis at nucleotides and amino acids level indicate that buffalo, cattle, sheep, pig and equine cathelicidin sequences comprise one clade which are distantly related with human, rabbit and murine cathelicidins. It may be reasonably concluded that buffalo possess the ancestral gene of cathelicidin like that of bovine species.     

Bovine and water buffalo <Emphasis Type="Italic">Mx2</Emphasis> genes: polymorphism and antiviral activity

Millennia-long selective pressure of single-strand RNA viruses on the bovine Mx locus has increased the advantages of using the bovine Mx protein to evaluate the ultimate significance of the antiviral role of Mx proteins. The conclusions of research based only on the bovine Mx1 protein showed the need for comprehensive studies that demonstrate the role of all isoforms, individually or together, especially in the presence of a second isoform, the bovine Mx2 gene. This study provides information about bovine and water buffalo Mx2 genes, as well as their allelic polymorphism and basic antiviral potential. Observation of an Mx2 cDNA sequence (2,381 bp) obtained from 15 animals from 11 breeds using primers based on a previous sequence (NCBI accession no. AF335147) revealed several nucleotide substitutions, with eight different alleles and two amino acid exchanges: Gly to Ser at position 302 and Ile to Val at position 354, though the latter was found only in the NCBI database. A water buffalo Mx2 cDNA sequence was identified for the first time, revealing 46 nucleotide substitutions with 12 amino acid variations, in addition to a 9-bp insertion in the 5′ untranslated region UTR, compared with the bovine Mx2 cDNA. Transfected 3T3 cells expressing bovine Mx2 mRNAs coding Gly or Ser at position 302, water buffalo Mx2 mRNA, positive control bovine Mx1 mRNA-expressing cells, and negative control parental 3T3 were subjected to infection with recombinant vesicular stomatitis virus (VSVΔG*-G), as were empty pCI-neo vector-transfected cells. The positive control and all cells expressing Mx2 mRNAs displayed significantly higher levels of antiviral activity against VSVΔG*-G (P < 0.01) than did the negative controls.     

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.     

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.     

Dog MHC alleles containing the human RA shared epitope confer susceptibility to canine rheumatoid arthritis

To determine whether canine rheumatoid arthritis (CRA) is associated with dog MHC (DLA-DRB1) alleles which contain the QRRAA/RKRAA conserved third hypervariable region (3HVR) sequence, DNA samples were extracted from 61 dogs with clinically diagnosed small-joint polyarthritis and from 425 controls. Breed-matched controls were available for 41 cases. DLA-DRB1 genotypes were identified using molecular typing methods. Phenotype frequencies were compared between cases and controls and odds ratios with 95% confidence intervals calculated. Several DLA-DRB1 alleles were associated with increased risk for CRA: DLA-DRB1*002, DRB1*009, and DRB1*018. This was also observed for the presence of any shared epitope (SE)-bearing allele. The associations with DLA-DRB1*002 and the SE were maintained when only breed-matched cases and controls were compared. This study suggests that a conserved amino acid motif in the 3HVR present in some DRB1 alleles of both dogs and humans is associated with rheumatoid arthritis in both species.     

Cross-reactive TCR responses to self antigens presented by different MHC class II molecules

Autoreactive T cells represent a natural repertoire of T cells in both diseased patients and healthy individuals. The mechanisms regulating the function of these autoreactive T cells are still unknown. Ob1A12 is a myelin basic protein (MBP)-reactive Th cell clone derived from a patient with relapsing-remitting multiple sclerosis. Mice transgenic for this human TCR and DRA and DRB1*1501 chains develop spontaneous experimental autoimmune encephalomyelitis. The reactivity of Ob1A12 is reported to be restricted to recognition of MBP peptide 85-99 in the context of DRB1*1501. DRA/DRB1*1501 and the patient's other restriction element, DRA/DRB1*0401, differ significantly in their amino acid sequences. In this study we describe an altered peptide ligand derived from MBP(85-99) with a single amino acid substitution at position 88 (Val to Lys; 88V-->K), that could stimulate the Ob1A12.TCR in the context of both DRA/DRB1*1501 and DRA/DRB1*0401. Analysis of a panel of transfected T cell hybridomas expressing Ob1A12.TCR and CD4 indicated that Ob1A12.TCR cross-reactivity in the context of DRA/DRB1*0401 is critically dependent on the presence of the CD4 coreceptor. Furthermore, we found that activation of Ob1A12.TCR with MBP altered peptide ligand 85-99 88V-->K presented by DRB1*1501 or DRB1*0401 resulted in significant differences in TCR zeta phosphorylation. Our data indicate that injection of altered peptide ligand into patients heterozygous for MHC class II molecules may result in unexpected cross-reactivities, leading to activation of autoreactive T cells.     

梅花鹿卵泡刺激素α-亚基cDNA的分子克隆与序列分析

从新屠宰的母梅花鹿脑垂体中提取总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的梅花鹿卵泡刺激素α-亚基CDNA片段,它将克隆至PMD-18-T-Verctor。随机挑选3个阳性重组子进行测序,并将测序结果与绵羊、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,梅花鹿卵泡刺激素α-亚基基因编码的氨基酸序列与绵羊、水牛的该基因同源性最高,达97%,只有4个氨基酸不同;与牛的该基因同源性达96%。与人的该基因氨基酸序列同源性较低,为75%。其编码的核苷酸序列与绵羊、水牛、牛的同源性最高,达96%,只有14-16个碱基不同,与人的基因核苷酸同源性最低,为84%,总的来说,哺乳动物的卵泡刺激素α-亚基具有很高的同源性。     

Biased T-cell receptor usage is associated with allelic variation in the MHC class II peptide binding groove

 A comprehensive analysis was carried out of the tri-molecular complex of peptide, major histocompatibility class II molecule, and T-cell receptor (TcR) involved in the recognition of the promiscuous HA (306–318) peptide, restricted by one of two closely related HLA-DR alleles, HLA-DRB1*0101 and HLA-DRB1*0103. These two DR molecules differ by only three amino acids at positions 67, 70, and 71, in the third variable region of the DRB1 chain. None of the HA (306–318)-specific T-cell clones restricted by these two DR molecules tolerated amino acid substitution at the peptide-binding position 71, despite the fact that the substitution did not interfere with peptide binding. The majority of the DRB1*0103-restricted clones tolerated substitution of the amino acid at the TcR-contacting position 70, while the DRB1*0101-restricted T cells did not. Biased usage of TRVA and TRVB segments was observed for the DRB1*0103-restricted clones; in contrast, apparently random usage was seen in the DRB1*0101-restricted T cells. Finally, limiting dilution analysis revealed a lower frequency of T cells reactive with the HA peptide in a DRB1*0103 compared with a DRB1*0101 individual. Taken together these data suggest that biased TcR gene usage may reflect a relatively low precursor frequency of T cells, and the need for clonal expansion of a limited set of high avidity T cells. Received: 7 August 1998 / Revised: 19 November 1998     

Identification of Cu/Zn superoxide dismutase in cattle and river buffaloes

All air-living organisms produce superoxide dismutase (SOD) and several antioxidant enzymes that limit oxidative stress by detoxifying reactive oxygen species. SOD1 gene has been investigated in Egyptian native cattle and buffalos at the level of genomic DNA and cDNA that were extracted from leucocytes. An unexpected band at approximately 370 bp was obtained in cattle genomic DNA and cDNA as well as in buffalo cDNA. SOD1 amplified sequence of native cattle genomic DNA and cDNA showed a 93% alignment. Native cattle genomic DNA SOD1 amplicon shares sequence homology with mRNAs of Bos taurus “similar to superoxide dismutase” (SOD1) sequence of the GeneBank database. It also shares sequence homology with “similar to superoxide dismutase” on B. taurus chromosome BTA13. The results indicate that the genomic DNA of Egyptian native cattle contains SOD1 processed pseudo gene. SOD1 primers amplified three fragments in buffalo genomic DNA which indicates that buffalo genome has different copies of SOD1 due to alternative splicing. It failed to produce the 370 bp fragments found in cattle DNA. The protein analysis revealed no differences between Egyptian native cattle and B. taurus SOD1 mRNA. However, one amino acid, aspartic acid (Asp), in Egyptian native cattle and B. taurus SOD1, is substituted with asparagine (Asn) (D26N) in buffaloes. This amino acid substitution may be due to non-synonymous single nucleotide polymorphisms (nsSNPs). The nsSNPs detected in buffaloes may affect the function of the encoded protein. This study is the first investigation reporting that the resistance of the buffalo to diseases and parasites that afflict cattle may not be acquired but may have a genetic basis.     

Evolution of the HLA-DR1 gene family. Structural and functional analysis of the new allele "DR-BON".

The molecules of the MHC are highly polymorphic and involve in Ag presentation; their striking genetic polymorphism allows probable interactions with a large variety of antigenic fragments when these are presented to the TCR. It is therefore of interest to explore the extent of this polymorphism and the mechanisms of its generation. We have studied the class II HLA-DR blank allele DR-BON that has been previously defined by MLR, restriction fragment length polymorphism, and two-dimensional gel electrophoresis. A cDNA library was constructed from a DR-BON homozygous typing cell and cDNA corresponding to DR alpha- and DR beta-chains were sequenced. By comparison with other known alpha- and beta-chain sequences it is shown that the alpha-chain is invariant and the beta-chain differs from DR1 by only six nucleotides, clustered in the third variable region with three amino acid changes at position 67, 70, and 71. The short DNA stretch of sequence encoding the 67-71 region is also present in other DR alleles: DR4/Dw10, DRw13, and some DRw11 specificities. Therefore we propose that a gene conversion-like event has occurred between the DRB1 *0101 (DR1/Dw1) gene and one of these three DRB1 genes. Extensive typing has been performed with a DR-BON-specific 17-mer oligonucleotide. Cross-hybridization with other genes than the ones expected from DNA sequence comparison was not observed. A selected panel of DR-BON reactive T cell clones shows three patterns of reactivity. Some clones are strictly DR-BON specific; some cross-reacted with DRw13 and a few cross-reacted with other haplotypes. The role of different epitopes of the third variable region of HLA-DR beta chain in allo-reaction is discussed.     

Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles

The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.     

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.     

Population genetic diversity of marble goby (<Emphasis Type="BoldItalic">Oxyeleotris marmoratus</Emphasis>) inferred from mitochondrial DNA and microsatellite analysis

Mastitis is an infectious disease of the mammary gland that leads to reduced milk production and change in milk composition. Complement component C3 plays a major role as a central molecule of the complement cascade involving in killing of microorganisms, either directly or in cooperation with phagocytic cells. C3 cDNA were isolated, from Egyptian buffalo and cattle, sequenced and characterized. The C3 cDNA sequences of buffalo and cattle consist of 5025 and 5019 bp, respectively. Buffalo and cattle C3 cDNAs share 99% of sequence identity with each other. The 4986 bp open reading frame in buffalo encodes a putative protein of 1661 amino acids—as in cattle—and includes all the functional domains. Further, analysis of the C3 cDNA sequences detected six novel single-nucleotide polymorphisms (SNPs) in buffalo and three novel SNPs in cattle. The association analysis of the detected SNPs with milk somatic cell score as an indicator of mastitis revealed that the most significant association in buffalo was found in the C >A substitution (ss: 1752816097) in exon 27, whereas in cattle it was in the C >T substitution (ss: 1752816085) in exon 12. Our findings provide preliminary information about the contribution of C3 polymorphisms to mastitis resistance in buffalo and cattle.