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.     

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.     

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.     

Rat sn-glycerol-3-phosphate acyltransferase: molecular cloning and characterization of the cDNA and expressed protein.

Rat mitochondrial glycerol-3-phosphate acyltransferase (GPAT) cDNA was cloned and characterized. We identified a cDNA containing an open reading frame of 828 amino acids that had an 89% homology with the coding region of the previously characterized mouse mitochondrial GPAT and a predicted amino acid sequence that was 96% identical. The rat 5' UTR was only 159 nucleotides, in contrast to the 926 nucleotide 5' UTR of the mouse cDNA and had an internal deletion of 167 nucleotides. GPAT was expressed in Sf21 insect cells, and specific inhibitors strongly suggest that, like the Escherichia coli GPAT, the recombinant mitochondrial GPAT and the mitochondrial GPAT isoform in rat liver contain critical serine, histidine, and arginine residues.     

Nucleotide sequence of cDNA clones coding for a brain-type fatty acid binding protein and its tissue-specific expression in adult zebrafish (Danio rerio)

We have determined the nucleotide sequence for two cDNA clones coding for a fatty acid binding protein (FABP) from zebrafish (Danio rerio). Comparison of the sequence with GenBank entries revealed extensive amino acid identity between this zebrafish FABP and brain FABPs (B-FABP) from other species. The zebrafish B-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases BglII or EcoRI suggesting that a single copy of the B-FABP gene is present in the zebrafish genome. Northern blot analysis demonstrated that the zebrafish B-FABP mRNA is approximately 850 nucleotides in length. In situ hybridization revealed that the B-FABP mRNA was expressed in the periventricular gray zone of the optic tectum of the adult zebrafish brain.     

Molecular cloning and sequencing of the banded dogfish (Triakis scyllia) interleukin-8 cDNA

The dogfish (Triakis scyllia) interleukin-8 (IL-8) cDNA was isolated from mitogen-stimulated peripheral white blood cells (WBCs) utilising the polymerase chain reaction (PCR). The cDNA sequence showed that the dogfish IL-8 clones contained an open reading frame encoding 101 amino acids. A short 5' untranslated region (UTR) of 70 nucleotides and a long 3' UTR of 893 nucleotides were also present in this 1.2-kb cDNA. Furthermore, the 3' UTR of the mRNA contained the AUUUA sequence that has been implicated in shortening of the half-life of several cytokines and growth factors. The predicted IL-8 peptide had one potential N-linked glycosylation site (Asn-72-Thr-74) that is not conserved in other vertebrates. It also contained four cysteine residues (Cys-34, 36, 61 and 77), which are characteristic of CXC subfamily cytokines and found in all vertebrates, to date. The dogfish IL-8 lacked an ELR motif as found in the lamprey and trout. Comparison of the deduced amino acids showed that the dogfish IL-8 sequence shared 50.5, 41.2, 37.1 and 40.4-45.5% identity with the chicken, lamprey, trout and mammalian IL-8 sequences, respectively.     

Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.     

Cloning and characterization of alpha(s2)-casein gene of Riverine buffalo.

The present study was carried out to characterize the alpha(s2)-casein gene in Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and alpha(s2)-casein cDNA were synthesized by RT-PCR, then cloned using pDRIVE-cloning vector and sequenced. The sequencing revealed that the size of alpha(s2)-casein was 669 bp with GC content of 41.11%. The gene encoded for 222 amino acid precursors and that it possessed 15 amino acids signal peptide. The similarity of buffalo alpha(s2)-casein mRNA sequence with that of cattle, sheep, goat, pig and camel were estimated as 97.9, 93.6, 93.4, 73.5 and 73.0%, respectively. In the phylogenetic trees, constructed from the data of the alpha(s2)-casein mRNA sequences as well as protein sequences, it has been observed that the cattle and buffalo were in the same group whereas sheep and goat formed another group. The camel and swine were placed in two separate groups.     

The sequence of a cloned cDNA for the beta subunit of bovine thyrotropin predicts a protein containing both NH2-and COOH-terminal extensions

Cloned cDNAs containing sequences coding for the beta subunit of bovine thyrotropin have been identified. The complete nucleotide sequence of the largest of the beta subunit cDNA inserts has been determined. This cDNA contains 35 nucleotides from the 5' untranslated region of thyrotropin beta subunit mRNA and 60 nucleotides coding for an NH2-terminal precursor segment. This is followed by 339 nucleotides which code for the published amino acid sequence of the thyrotropin beta subunit. Following the 339 nucleotide beta subunit coding sequence, no termination codon is encountered for another 15 nucleotides. Thus, the cDNA codes for a thyrotropin beta subunit containing an additional 5 amino acids at the COOH terminus. The cDNA also contains 82 nucleotides of 3' untranslated sequence followed by a short poly(A) segment. Comparison of the bovine cDNA sequence to the recently described mouse thyrotropin beta subunit cDNA sequence reveals considerable homology throughout the coding sequence, including the COOH-terminal extension. These findings suggest the possibility that a thyrotropin beta subunit precursor is processed at both the NH2 and COOH termini.     

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.     

Nucleotide sequence of hamster S-adenosylmethionine decarboxylase cDNA.

The nucleotide sequence of a cDNA encoding the proenzyme of hamster S-adenosylmethionine decarboxylase including 169 nucleotides of the 5' untranslated region has been determined. The deduced amino acid sequence shows a remarkable similarity to the human proenzyme with only seven differences out of 334 amino acids. The nucleotide sequence of the 5' untranslated region showed 93% homology with the corresponding rat and human sequences suggesting that this region may play an important role in the regulation of S-adenosylmethionine decarboxylase expression.     

Complete nucleotide sequence of ovine alpha-lactalbumin mRNA

The nucleotide sequence of ovine alpha-lactalbumin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and a primer extension product. Ovine alpha-lactalbumin mRNA contains 723 nucleotides (excluding the poly(A) tail), with a 5' non-coding region of 26 nucleotides, followed by the 426 nucleotides of the coding region which determines a sequence signal of 19 amino acid residues and the 123 amino acid residues of mature alpha-lactalbumin. The coding region is followed by a 3' untranslated sequence of 271 nucleotides. The derived amino acid sequence of ovine pre-alpha-lactalbumin differs from that of its bovine counterpart by 8 amino acid substitutions, all but one originating from single mutations. Comparison of sequences of guinea pig, rat and human alpha-lactalbumin mRNAs with their ovine and bovine counterparts has revealed that these molecules have rapidly evolved. The highest degree of conservation was observed in the region coding for the mature protein and corresponds essentially to sequences which interact with UDP-galactosyltransferase and Ca2+ ions.     

Fatty-acid-binding protein from bovine brain. Amino acid sequence and some properties

A fatty-acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified FABP behaved as an anionic protein with an apparent molecular mass of 14.7 kDa; its complete amino acid sequence was determined and microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP and bovine mammary-derived growth inhibitor (MDGI).     

Cloning and Characterization of Full-length Triticin cDNA and Genes from Wheat Varieties K-68 and Chinese Spring

Full-length cDNA and genes for triticin protein were cloned and characterized from wheat varieties K-68 and Chinese Spring differing considerably in grain colour, total protein content, grain hardness, milling behaviour and baking characteristics. Wheat variety K-68 possesses excellent chapatti (unleavened bread) making quality in contrast to Chinese Spring. The nucleotide and deduced amino acid sequences of the full-length triticin cDNA and genes were compared with those of other legumin genes. Although minor variations in the nucleotide sequences were observed when compared with the published sequence of the partial triticin cDNA clone λTri-25, the deduced amino acid sequence of the full-length triticin cDNA clone (Tri-cK68) revealed large variation in the Hyper Variable Region. The deduced amino acid sequence of the full-length triticin cDNA clone Tri-cK68 revealed two lysine-rich regions in the triticin protein. Comparative analysis of the nucleotide sequences of the triticin genes with the cDNA clone λTri-25 revealed the presence of a stretch of 31 nucleotides in the 5′ UTR of λTri-25 having exact complementarity with a stretch of nucleotides of the same length in the 3′ UTR of the full length triticin genes cloned from the wheat varieties K-68 (Tri-gK68) and Chinese Spring (Tri-gCS). Analysis of the nucleotide sequence of triticin promoter (Tri-pK68) revealed the presence of several elements responsible for seed-specific expression and responsiveness to light.     

猪CuZnSOD基因的克隆、表达及功能分析

为了进一步了解和认识CuZnSOD基因的结构和功能,揭示CuZnSOD对猪抗氧化机能的影响,寻找与肉质性状相关联的分子标记,文章采用RACE(Rapid amplification of cDNA end)方法,对莱芜猪CuZnSOD基因cDNA进行克隆测序,分析其结构和功能,并用Real-timePCR检测CuZnSOD基因的表达.结果表明,CuZnSOD基因cDNA序列全长658 bp(GenBank登录号:GU944822),包含76 bp的5'UTR和120 bp的3'UTR序列.全部CDS序列462 bp,编码153个氨基酸,分子量为15.9 kDa,等电点为6.03.CuZnSOD基因编码的氨基酸序列中,第3氨基酸残基处存在1个O-糖基化位点,第86氨基酸残基处存在1个N-糖基化位点.二级结构中α螺旋仅占1.31%.在进化过程中高度保守,与人、牛、小鼠和褐鼠的编码区同源性分别为87.74%、87.66%、83.44%和83.23%;氨基酸序列同源性分别为90.26%、94.12%、92.21%和91.50%.CuZnSOD存在典型的金属结合配体结构域(GFHVHQFGDNT).基于蛋白序列所构建的分子进化树表明猪与牛的亲缘关系最近.在mRNA水平上,CuZnSOD是一个广谱表达基因,在大脑、心脏、脾脏、肝脏、肾脏、肺、大肠、小肠、脊髓,肌肉、背膘和胃中都能检测到,其在肾脏,小肠和肺中表达量较高,在心脏和肌肉组织中表达量较低.