Cloning and expression of caprine KIT gene and associations of polymorphisms with litter size

2016 ◽  
Vol 56 (10) ◽  
pp. 1579 ◽  
Author(s):  
X. P. An ◽  
J. X. Hou ◽  
T. Y. Gao ◽  
B. Y. Cao
Keyword(s):  
Amino Acid ◽  
Litter Size ◽  
Bos Taurus ◽  
Homo Sapiens ◽  
Ovis Aries ◽  
Cloning And Expression ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Reading Frame ◽  
Kit Gene

The full coding region of KIT mRNA was cloned from the caprine ovary. The results showed the caprine KIT cDNA (GenBank accession number KF364483) contained a 2925-bp open reading frame encoding a protein with 974 amino acid residues. BLAST analysis revealed that the caprine KIT protein had high similarity with that of four species: Ovis aries (99%), Bos taurus (99%), Sus scrofa (94%) and Homo sapiens (90%). The KIT mRNA expression pattern showed that KIT mRNA was expressed highly in kidney, ovary, uterus and breast. Two single nucleotide polymorphisms (g.88430T > A and g.120466G > A) in the caprine KIT gene were detected by PCR–restriction fragment length polymorphism (RFLP) and DNA sequencing in 735 goats of Xinong Saanen, Guanzhong and Boer breeds. The g.88430T > A mutation was a missense mutation (Tyr > Asn at position 409 amino acid of KIT). The association study has been done by jointly analysing all data in one analysis. The result showed that individuals with TT and TA genotypes had their litter size increased by 0.11 and 0.09, respectively, compared with those with AA genotype at the g.88430T > A locus for three goat breeds (P < 0.05). Further analysis revealed that combined genotype TTAA was better than the others for litter size in three goat breeds. Therefore, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the KIT gene could serve as a genetic marker for litter size in goat breeding.

scholarly journals Polymorphism Detection of GDF9 Gene and Its Association with Litter Size in Luzhong Mutton Sheep (Ovis aries)

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 571
Author(s):  
Fengyan Wang ◽  
Mingxing Chu ◽  
Linxiang Pan ◽  
Xiangyu Wang ◽  
Xiaoyun He ◽  
...  
Keyword(s):  
Litter Size ◽  
Tertiary Structure ◽  
Novel Mutation ◽  
Major Gene ◽  
Ovis Aries ◽  
Nucleotide Polymorphisms ◽  
Economic Traits ◽  
Coding Region ◽  
Association Analyses ◽  
Gdf9 Gene

Litter size is one of the most important economic traits in sheep. GDF9 and BMPR1B are major genes affecting the litter size of sheep. In this study, the whole coding region of GDF9 was sequenced and all the SNPs (single nucleotide polymorphisms) were determined in Luzhong mutton ewes. The FecB mutation was genotyped using the Sequenom MassARRAY®SNP assay technology. Then, the association analyses between polymorphic loci of GDF9 gene, FecB, and litter size were performed using a general linear model procedure. The results showed that eight SNPs were detected in GDF9 of Luzhong mutton sheep, including one novel mutation (g.41769606 T > G). The g.41768501A > G, g.41768485 G > A in GDF9 and FecB were significantly associated with litter size in Luzhong mutton ewes. The g.41768485 G > A is a missense mutation in the mature GDF9 protein region and is predicted to affect the tertiary structure of the protein. The results preliminarily demonstrated that GDF9 was a major gene affecting the fecundity of Luzhong mutton sheep and the two loci g.41768501A > G and g.41768485 G > A may be potential genetic markers for improving litter size.

scholarly journals IMGT® Biocuration and Comparative Analysis of Bos taurus and Ovis aries TRA/TRD Loci

Genes ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 30
Author(s):  
Perrine Pégorier ◽  
Morgane Bertignac ◽  
Viviane Nguefack Ngoune ◽  
Géraldine Folch ◽  
Joumana Jabado-Michaloud ◽  
...  
Keyword(s):  
Immune Response ◽  
Comparative Analysis ◽  
T Cell ◽  
T Cell Receptors ◽  
Bos Taurus ◽  
Homo Sapiens ◽  
Adaptive Immune Response ◽  
Ovis Aries ◽  
Cell Receptors ◽  
Adaptive Immune

The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens to generate immunity, and mount stronger attacks each time the pathogen is encountered. T cell receptors are the antigen receptors of the adaptive immune response expressed by T cells, which specifically recognize processed antigens, presented as peptides by the highly polymorphic major histocompatibility (MH) proteins. T cell receptors (TR) are divided into two groups, αβ and γδ, which express distinct TR containing either α and β, or γ and δ chains, respectively. The TRα locus (TRA) and TRδ locus (TRD) of bovine (Bos taurus) and the sheep (Ovis aries) have recently been described and annotated by IMGT® biocurators. The aim of the present study is to present the results of the biocuration and to compare the genes of the TRA/TRD loci among these ruminant species based on the Homo sapiens repertoire. The comparative analysis shows similarities but also differences, including the fact that these two species have a TRA/TRD locus about three times larger than that of humans and therefore have many more genes which may demonstrate duplications and/or deletions during evolution.

scholarly journals Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

2000 ◽  
Vol 68 (7) ◽  
pp. 3941-3948 ◽  
Author(s):  
Tong-Soo Kim ◽  
Younghun Jung ◽  
Byoung-Kuk Na ◽  
Ki-Sun Kim ◽  
Pyung-Rim Chung
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Fasciola Hepatica ◽  
Human Subjects ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Gene Fragment ◽  
Coding Region ◽  
Sod Activity ◽  
Degenerate Oligonucleotide

ABSTRACT The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

scholarly journals Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase, an Enzyme Involved in the Metabolism of Daidzein, from Equol-Producing Lactococcus Strain 20-92

2010 ◽  
Vol 76 (17) ◽  
pp. 5892-5901 ◽  
Author(s):  
Yoshikazu Shimada ◽  
Setsuko Yasuda ◽  
Masayuki Takahashi ◽  
Takashi Hayashi ◽  
Norihiro Miyazawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enteric Bacteria ◽  
Amino Acid Sequences ◽  
Soy Isoflavones ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Binding Motifs ◽  
Reading Frame ◽  
Cofactor Binding ◽  
International Patent

ABSTRACT Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

scholarly journals Identification, Cloning, and Expression of the CAMP-Like Factor Autotransporter Gene (cfa) of Bartonella henselae

2005 ◽  
Vol 73 (7) ◽  
pp. 4205-4213 ◽  
Author(s):  
Christine M. Litwin ◽  
Joel M. Johnson
Keyword(s):  
Amino Acid ◽  
Bartonella Henselae ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cloning And Expression ◽  
Cosmid Library ◽  
Group B Streptococci ◽  
Reading Frame ◽  
Calculated Molecular Weight ◽  
The Family

ABSTRACT The CAMP reaction was first described by Christie et al. (R. Christie, N. E. Atkins, and E. Munch-Petersen, Aust. J. Exp. Biol. 22:197-200, 1944) as the synergistic lysis of sheep red blood cells by Staphylococcus aureus sphingomyelinase and CAMP factor (cohemolysin), a secreted protein from group B streptococci. We observed a CAMP-like reaction when Bartonella henselae was grown in close proximity to S. aureus on 5% sheep blood agar. This study describes the cloning, sequencing, and characterization of a CAMP-like factor autotransporter gene (cfa) from B. henselae. A cosmid library of B. henselae ATCC 49793 was constructed using SuperCos1 in Escherichia coli XL1-Blue MR. Cosmids were screened for the CAMP reaction, and a quantitative cohemolysis microtiter assay was developed using purified sphingomyelinase. Cosmid clones with the strongest cohemolytic reaction had similar restriction enzyme patterns. A DNA fragment that expressed the cohemolysin determinant was subcloned in a 7,200-bp StuI-BamHI fragment which contained a 6,024-bp open reading frame. The deduced amino acid sequence showed homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane. They contain an N-terminal passenger region, the α-domain, and a C-terminal transporter region, the β-domain. The α-domain contained four, nearly identical 42-amino-acid repeats and showed homology to the family of RTX (repeat in toxin) hemolysins. The concentrated supernatant of the recombinant strain expressed a protein with a molecular mass of 180 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis consistent with the calculated molecular weight of the secreted α-domain. In conclusion, we have characterized a novel secreted cohemolysin autotransporter protein of B. henselae.

scholarly journals Evidence for Production of a New Lantibiotic (Butyrivibriocin OR79A) by the Ruminal Anaerobe Butyrivibrio fibrisolvensOR79: Characterization of the Structural Gene Encoding Butyrivibriocin OR79A

1999 ◽  
Vol 65 (5) ◽  
pp. 2128-2135 ◽  
Author(s):  
M. L. Kalmokoff ◽  
D. Lu ◽  
M. F. Whitford ◽  
R. M. Teather
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Northern Blotting ◽  
Coding Region ◽  
Gene Encoding ◽  
Butyrivibrio Fibrisolvens ◽  
Reading Frame ◽  
Significant Homology

ABSTRACT The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79Awas a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5′ region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.

scholarly journals Cloning and Expression Analysis of a Farnesyl Diphosphate Synthase (FPPS) Gene from Chamaemelum nobile

2017 ◽  
Vol 45 (2) ◽  
pp. 358-364 ◽  
Author(s):  
Xiao-Meng LIU ◽  
Ting-Ting TAO ◽  
Xiang-Xiang MENG ◽  
Wei-Wei ZHANG ◽  
Jie CHANG ◽  
...  
Keyword(s):  
Amino Acid ◽  
Condensation Reaction ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Farnesyl Diphosphate ◽  
Cloning And Expression ◽  
Farnesyl Diphosphate Synthase ◽  
Reading Frame ◽  
Chamaemelum Nobile ◽  
Multiple Alignment Analysis

Farnesyl diphosphate synthase (FPPS), an isopentenyl transferase, catalyzes the condensation reaction of five carbon isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form fifteen carbon farnesyl pyrophosphate (FPP), which is the key precursor for sesquiterpene biosynthesis. In this study, a FPPS gene (CnFPPS) was cloned from Chamaemelum nobile. The full-length cDNA of CnFPPS is 1239 bp and contains an open reading frame (ORF) of 1029 bp encoding 342 amino acids. The theoretical molecular weight and pI of the CnFPPS protein are 39.38 kDa and 5.59, respectively. Multiple alignment analysis showed the protein sequence of CnFPPS had a high homology with FPPS proteins from other plants. The deduced amino acid of CnFPPS contained five conservative domains such as substrate binding pocket, substrate-Mg2+ binding site, catalytic site, aspartate-rich region 1 and 2, suggesting CnFPPS is one member of FPPS family in C. nobile. Phylogenetic analysis based on the amino acid sequences of FPPSs showed that CnFPPS was closely related to the FPPS of Matricaria chamomilla. The result of qRT-PCR revealed that CnFPPS gene was constitutively expressed in different tissues of C. nobile, with the highest expression in the root. These findings improve the understanding of the synthesis and regulation of the terpenoid compounds at the molecular level and lay a foundation for studying the regulatory functions of CnFPPS in terpenoid biosynthetic pathway in C. nobile.

scholarly journals Dipeptidyl peptidase III is a zinc metallo-exopeptidase: Molecular cloning and expression

1998 ◽  
Vol 329 (2) ◽  
pp. 275-282 ◽  
Author(s):  
Katsuhiko FUKASAWA ◽  
M. Kayoko FUKASAWA ◽  
Makoto KANAI ◽  
Shingo FUJII ◽  
Junzo HIROSE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dipeptidyl Peptidase ◽  
Cloning And Expression ◽  
Zinc Binding ◽  
Binding Motif ◽  
Ph Optimum ◽  
Reading Frame ◽  
Theoretical Molecular Mass ◽  
Cloned Cdna ◽  
Zinc Binding Domain

We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta. It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-β-naphthylamide. Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-β-naphthylamides were not hydrolysed at all. The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme. The zinc dissociation constant was 250 fM at pH 7.4 as determined by the zinc binding study. We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme. The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da. Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-β-naphthylamide. The recombinant protein was purified and the amino acid sequence of the protein was confirmed. We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme. It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases.

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