Molecular characterization of cDNA encoding B. taurus cathelicidin-7 like antibiotic peptide from bone marrow cells of Bubalus bubalis.

Cathelicidins represent a diverse family of endogenous cationic antibiotic peptide present in all mammalian species. In the present study, a novel cathelicidin cDNA was identified and characterized from bone marrow cells of buffalo (Bubalus bubalis) using RT-PCR based approach. The cDNA encodes a propeptide of 1.18 kDa with net positive charge at neutral pH. The precursor peptide possesses a signal peptide of 29 amino acids and a biologically active peptide of 34 residues. Comparison of sequences indicates only 66.1 and 64.1% identity at nucleotides and amino acids level respectively, with the already reported cathelicidin congener from the same species. However, high degree of similarity (92.8% nucleotides and 81.9% amino acids) was noticed with cathelicidin 7 sequence of Bos taurus suggesting interspecies conservation of cathelicidin peptides rather than intra-species within bovidae family. Phylogenetic trees analyses also support these data. Our findings, further justify the cloned cDNA as a unique cathelicidin member of B. bubalis, and may reasonably considered to be another example of structural diversity exhibited by cathelicidin-derived peptides as reported from other mammals.     

Detection of genome specific monomorphic loci in Bos taurus and Bubalus bubalis with oligodeoxyribonucleotide probe

A synthetic oligodeoxyribonucleotide probe (OAT36) comprising nine repeats of 5'GACA 3′ and several enzymes were used to analyse cow, (Bos taurus) and buffalo (Bubalus bubalis) genomes and a number of monomorphic loci were detected in both the species. Different animals from the same species showed an almost ‘similar’ monomorphic hybridization pattern but animals from two separate species showed a different ‘genome specific’ pattern. The overall hybridization with any enzyme and probe combination was found to be unique to one species. This forms the basis of genome specific hybridization which is substantiated by our zoo-blot hybridization studies. The evolutionary aspect of these loci in the context of sequence polymorphisms is discussed.     

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β(1) domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.     

Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat

In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo-tolerant as development proceeds. Additionally, the resistance to heat shock was a result of the genetic contribution from both oocyte and spermatozoa.     

Isolation and characterization of the Bos taurus beta-casein gene

The expression of casein genes in the mammary cells is regulated by peptide and steroid hormones. To investigate the controlling mechanisms we have isolated and characterized the bovine beta-casein gene. The gene has the size of 8.6 kb, which is 7.8 times longer than the corresponding mRNA composed of nine exons. The genomic clones include additional 8.5-kb and 4.5-kb sequences of the 5'- and 3'-flanking regions. We have determined the sequences of the 5' and 3' ends of the gene and compared them with the respective sequences of the rat beta-casein gene. Conserved sequences are identical or homologous to the potential binding sites for nuclear factors and for glucocorticoid and progesterone receptors. The regulatory region contains two different TATA signals and a repeat sequence between them.     

Molecular cloning and characterization of beta-defensin cDNA expressed in distal ileum of buffalo (Bubalus bubalis).

Defensins play a prominent role in protection of various epithelial surfaces. In this study, we have cloned and characterized the mRNA from the distal ileum of Bubalus bubalis. Total RNA after isolation from ileal epithelium was reverse transcribed to synthesize cDNA using primers designed by taking conserved region of cattle enteric beta-defensin (EBD) mRNA, goat beta-defensin 2 (BD 2) and cattle lingual antimicrobial peptide (LAP) mRNA sequences. The PCR amplified cDNA of 254 bp was ligated to pDrive cloning vector and transformed into XL-blue strain of E coli. The sequence analysis indicated 29 nucleotide substitutions with reported cattle EBD mRNA sequence sharing 86.2% homology, 92.1% with cattle LAP, 81.6% with cattle tracheal antimicrobial peptide and 84.6% with goat BD 2. The deduced amino acid sequence encodes for a 64 amino acid precursor peptide. Both nucleotide and amino acid sequence homology shows that the cloned sequence is closer to cattle LAP.     

Response of prepubertal Bos taurus and Bos indicus x Bos taurus heifers to melengestrol acetate with or without gonadotropin-releasing hormone

The effectiveness of treatments to induce estrus in prepubertal beef heifers was evaluated. Angus x Hereford (n = 148) and Brahman x Hereford (n = 148) heifers were sorted after weaning by body weight into light and heavy weight blocks. Heifers were assigned to diets, calculated to reach a target weight of 55% or 65% of their projected mature weight by the start of breeding. Cyclicity was determined after a 160-d observation period and from concentrations of progesterone in serum determined 10 d before and on the day that treatments began to induce puberty. The remaining nonpubertal heifers, with concentrations of progesterone in serum of less than 1 ng/ml (0 or 10 d before treatment), were assigned randomly within breed and nutrition group to either a melengestrol acetate + saline (MGA+S) or MGA + gonadotropin-releasing hormone (MGA+GnRH) treatment. Prepubertal Angus x Hereford heifers (n = 11) and Brahman x Hereford heifers (n = 49) were fed 0.5 mg MGA for 7 d. Forty-eight hours after MGA, heifers were injected with 500 ug s.c. GnRH or 5 ml of saline. Blood samples were collected from all prepubertal heifers every 3 d after GnRH or saline for 30 d. There was no difference between treatments in the proportion of heifers that exhibited estrus by Day 7 after treatment. However, a larger (P<0.05) proportion of MGA+S-treated heifers exhibited estrus within 14 d after treatment than MGA+GnRH-treated heifers (87 vs 63%). Among heifers that exhibited estrus during that time period, the proportion with increased progesterone was higher (P<0.10) for the MGA+GnRH group than for the MGA+S group (71 vs 41%, Day 7; 79 vs 54%, Day 14). There was no difference in conception rate at first service between treatment groups. Thirty-seven and 53%, respectively, of the MGA+S and MGA+GnRH-treated heifers had short estrous cycles after treatment, and 44 and 50%, respectively, of those short cycles were repeated. Pregnancy rates at the end of 45 d were numerically higher for MGA+S heifers than for MGA+GnRH treated counterparts (63 vs 53%).     

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.     

The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.     

Nucleotide sequence and variation of IGF2 gene exon 6 in Bos taurus and Bos indicus cattle

The major assumption of this study is that polymorphism of a gene could be used to investigate its allele-specific expression as well as its methylation and imprinting status. Therefore, the aim of this study was to analyze the polymorphism of the coding region of the bovine IGF2 gene and to determine the sequence of its gene exon 6 in Bos taurus and Bos indicus cattle. A single nucleotide "C" deletion/insertion polymorphism was found in both cattle subspecies and a G/T transversion (RFLP-MboII) in the Bos indicus IGF2 gene. A 407-bp fragment of bovine IGF2 exon 6 was sequenced and the sequences (including variable nucleotides) were deposited in the GenBank database. A comparative analysis was performed for this fragment from different species; 99.5% identity was found between Bos taurus and Bos indicus cattle.     

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts.

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function.     

A novel endothelial tyrosine kinase cDNA homologous to platelet-derived growth factor receptor cDNA.

Degenerate oligonucleotide primers complementary to the highly conserved subdomains III and VIII of subclass III tyrosine kinase receptors (TKr-III) were utilized to amplify rat aortic cDNA by polymerase chain reaction. Most of the cloned DNA products were rat platelet-derived growth factor receptor beta and macrophage-colony stimulating growth factor receptor cDNAs. Screening of the clones with probes coding for the receptor-specific kinase insert domain allowed the identification of a novel putative TKr-III cDNA, which hybridized with a approximately 6.1 kb mRNA with a distinctive tissue distribution. In situ hybridization on rat tissues and Northern analysis of cultured cells indicate that endothelial cells express a novel putative TKr-III mRNA.     

The complete coding region sequence of river buffalo (Bubalus bubalis) SRY gene.

The Y-linked SRY gene is responsible for testis determination in mammals. Mutations in this gene can lead to XY Gonadal Dysgenesis, an abnormal sexual phenotype described in humans, cattle, horses and river buffalo. We report here the complete river buffalo SRY sequence in order to enable the genetic diagnosis of this disease. The SRY sequence was also used to confirm the evolutionary divergence time between cattle and river buffalo 10 million years ago.     

Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor.

A novel gene coding for the pre-B-cell colony-enhancing factor (PBEF) has been isolated from a human peripheral blood lymphocyte cDNA library. The expression of this gene is induced by pokeweed mitogen and superinduced by cycloheximide. It is also induced in the T-lymphoblastoid cell line HUT 78 after phorbol ester (phorbol myristate acetate) treatment. The predominant mRNA for PBEF is approximately 2.4 kb long and codes for a 52-kDa secreted protein. The 3' untranslated region of the mRNA has multiple TATT motifs, usually found in cytokine and oncogene messages. The PBEF gene is mainly transcribed in human bone marrow, liver tissue, and muscle. We have expressed PBEF in COS 7 and PA317 cells and have tested the biological activities of the conditioned medium as well as the antibody-purified protein in different in vitro assays. PBEF itself had no activity but synergized the pre-B-cell colony formation activity of stem cell factor and interleukin 7. In the presence of PBEF, the number of pre-B-cell colonies was increased by at least 70% above the amount stimulated by stem cell factor plus interleukin 7. No effect of PBEF was found with cells of myeloid or erythroid lineages. These data define PBEF as a novel cytokine which acts on early B-lineage precursor cells.