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Sample records for molecular cloning tissue

  1. Molecular cloning, tissue distribution, and immune function of goose TLR7.

    PubMed

    Qi, Yulin; Chen, Shun; Zhao, Qiurong; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-02-01

    TLR7 is a transmembrane endosomal protein that plays an essential role in innate antiviral responses via the recognition of conserved viral molecular patterns. Here, we cloned the full-length cDNA of goose TLR7 and carried out a molecular characterization of goose TLR7. The goose TLR7 gene is 3900 bp and encodes a 1045 amino acid protein with high homology to poultry (93% to duck and 83% to chicken). Similar conclusions were made by phylogenetic analysis. The predicted protein secondary structure of goose TLR7 contained a conserved Toll/interleukin-1 receptor domain and characteristic leucine-rich repeat regions, which has also been reported for duck TLR7. Additionally, the tissue distribution of goose TLR7 suggests that immune-associated tissues, especially the cecal tonsil and bursa of Fabricius, have high goose TLR7 expression levels. Goose TLR7 is abundantly expressed in lung tissues, which is distinct from its expression in chickens. Similar to duck TLR7, goose spleen mononuclear cells (MNCs) exposed to the mammalian TLR7 agonists R848 and Imiquimod showed significant induction of the production of proinflammatory cytokines and IFN-α. New type gosling viral enteritis virus (NGVEV) infection resulted in high mRNA expression levels of goose TLR7 in the spleen. By contrast, no direct interaction between NGVEV and goose TLR7 was detected after infecting goose spleen MNCs with NGVEV in vitro. However, triggering of goose TLR7 resulted in the rapid up-regulation of proinflammatory cytokines and anti-viral molecules, suggesting that goose TLR7 plays an important role in anti-viral defense.

  2. Molecular cloning and synthesis of biologically active human tissue inhibitor of metalloproteinases in yeast

    SciTech Connect

    Kaczorek, M.; Honore, N.; Ribes, V.; Dehoux, P.; Cornet, P.; Cartwright, T.; Streeck, R.E.

    1987-06-01

    Tissue inhibitor of metalloproteinases (TIMP) is a widely distributed glycoprotein that stochiometrically inactivates metalloproteinases involved in connective tissue catabolism. Here they report the cDNA cloning of TIMP from human fibroblastic MRC5 cells using a single 42-base oligonucleotide probe. Expression in S. cerevisiae of complete TIMP cDNA yielded insoluble protein aggregates. Biologically active TIMP was reconstituted from the yeast product by a denaturation/renaturation procedure.

  3. Molecular cloning, sequence analysis and tissue-specific expression of Akirin2 gene in Tianfu goat.

    PubMed

    Ma, Jisi; Xu, Gangyi; Wan, Lu; Wang, Nianlu

    2015-01-01

    The Akirin2 gene is a nuclear factor and is considered as a potential functional candidate gene for meat quality. To better understand the structures and functions of Akirin2 gene, the cDNA of the Tianfu goat Akirin2 gene was cloned. Sequence analysis showed that the Tianfu goat Akirin2 cDNA full coding sequence (CDS) contains 579bp nucleotides that encode 192 amino acids. A phylogenic tree of the Akirin2 protein sequence from the Tianfu goat and other species revealed that the Tianfu goat Akirin2 was closely related with cattle and sheep Akirin2. RT-qPCR analysis showed that Akirin2 was expressed in the myocardium, liver, spleen, lung, kidney, leg muscle, abdominal muscle and the longissimus dorsi muscle. Especially, high expression levels of Akirin2 were detected in the spleen, lung, and kidney whereas lower expression levels were seen in the liver, myocardium, leg muscle, abdominal muscle and longissimus dorsi muscle. Temporal mRNA expression showed that Akirin2 expression levels in the longissimus dorsi muscle, first increased then decreased from day 1 to month 12. Western blotting results showed that the Akirin2 protein was only detected in the lung and three skeletal muscle tissues.

  4. Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue.

    PubMed

    Jiang, Ming Feng; Hu, Ming Jun; Ren, Hong Hui; Wang, Li

    2015-12-01

    Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

  5. A novel tissue inhibitor of metalloproteinase in blood clam Tegillarca granosa: molecular cloning, tissue distribution and expression analysis.

    PubMed

    Wang, Qing; Bao, Yongbo; Huo, Lihui; Gu, Hailong; Lin, Zhihua

    2012-09-01

    Tissue inhibitor of metalloproteinases (TIMPs) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has been found to be broader as it includes the inhibition of several of the MMPs, etc. The cDNA encoding TIMP-4-like gene from blood clam Tegillarca granosa (designated as Tg-TIMP-4-like) which is the first tissue inhibitor of metalloproteinase identified in blood clams, was cloned and characterized. It was of 1164 bp, and an open reading frame (ORF) of 666 bp encoding a putative protein of 222 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other TIMP homologues and showed the highest (30.56%) identity to the TIMP-1.3 from Crassostrea gigas. Several highly conserved motifs including several TIMP signatures, amino acid residue Cys³⁰ responsible for coordinating the metal ions, the Cys-X-Cys motif and the putative NTR (netrin) domain were almost completely conserved in the deduced amino acid of Tg-TIMP-4 like, which indicated that Tg-TIMP-4-like should be a member of the TIMP family. The mRNA expression of Tg-TIMP-4-like in the tissues of mantle, adductor muscle, foot, gill, hemocyte and hepatopancreas was examined by quantitative real-time PCR (qT-PCR) and mRNA transcripts of Tg-TIMP-4-like were mainly detected in hemocyte, and weakly detected in the other tissues. We also observed that Tg-TIMP-4 like mRNA accumulated significantly during Vibrio parahaemolyticus, Peptidogylcan (PGN) and Lipopolysaccharide (LPS) challenge, whereas the timing and quantitative differences of mRNA expression against different challenge indicated that Tg-TIMP-4-like may play a pivotal role in mollusc defense mechanisms. PMID:22771965

  6. Molecular cloning, structural analysis, and tissue expression of the TNNT3 gene in Guizhou black goat.

    PubMed

    Chen, Haolin; Zhang, Jinhua; Yu, Bo; Li, Liang; Shang, Yishun

    2015-11-15

    The vertebrate fast skeletal troponin T (TNNT3) protein is an important regulatory and structural component of thin filaments in skeletal muscle, which improves meat quality traits of livestock and poultry. In this study, the troponin T isoforms from adult goat (skeletal muscle mRNA) were identified. We isolated the full-length coding sequence of the goat TNNT3 gene (GenBank: KM042888), analyzed its structure, and investigated its expression in different tissues from different aged goats (10, 30, 90, 180, and 360 days old). Real-time quantitative reverse transcription-polymerase chain reaction analyses revealed that Guizhou black goat TNNT3 was highly expressed in the biceps femoris muscle, abdominal muscle, and longissimus dorsi muscle (P<0.01), and lowly expressed in the cardiac muscle, masseter muscle, and rumen tissue (P>0.05). Western blotting confirmed that the TNNT3 protein was expressed in the muscle tissues listed above, with the highest level found in the longissimus dorsi muscle, and the lowest level in the masseter muscle. In the 10 to 360day study period the TNNT3 protein expression level was the highest when the goats were 30 days old. A peptide, ASPPPAEVPEVHEEVH that may contribute to improved goat meat tenderness was identified. This study provides an insight into the molecular structure of the vertebrate TNNT3 gene.

  7. Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in spotted scat, Scatophagus argus.

    PubMed

    Li, Jian-Tao; Yang, Zhao; Chen, Hua-Pu; Zhu, Chun-Hua; Deng, Si-Ping; Li, Guang-Li; Tao, Ya-Xiong

    2016-05-01

    Melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure in mammals. The functions of the MC4R in fish have not been investigated extensively. We herein reported on the cloning, tissue distribution, and pharmacological characterization of spotted scat (Scatophagus argus) MC4R (SAMC4R). It consisted of a 984bp open reading frame predicted to encode a protein of 327 amino acids. Sequence analysis revealed that SAMC4R was highly homologous (>80%) at amino acid levels to several teleost MC4Rs. Phylogenetic analyses showed that SAMC4R was closely related to piscine MC4R. Using RT-PCR, we showed that in addition to brain, pituitary, and gonads, mc4r mRNA was also widely expressed in peripheral tissues of spotted scat in sexually divergent pattern. With human MC4R (hMC4R) as a control, several agonists including α-melanocyte stimulating hormone (α-MSH), [Nle(4), D-Phe(7)]-α-MSH (NDP-MSH), adrenocorticotropic hormone (ACTH) and THIQ (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine), were used to investigate the binding and signaling properties of SAMC4R. The results showed that SAMC4R bound NDP-MSH with the highest affinity followed by ACTH (1-24) and α-MSH. Similar ranking was also found for hMC4R, although SAMC4R had two to five-fold higher affinities for these ligands. THIQ did not displace NDP-MSH from SAMC4R, different from hMC4R. α-MSH, NDP-MSH, and ACTH (1-24) were identified as potent agonists to stimulate cAMP generation followed by THIQ in SAMC4R. The availability of SAMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in spotted scat.

  8. Type I interferon receptors in goose: molecular cloning, structural identification, evolutionary analysis and age-related tissue expression profile.

    PubMed

    Zhou, Hao; Chen, Shun; Qi, Yulin; Zhou, Qin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-04-25

    The cDNAs encoding two distinct type I interferon receptors were firstly cloned from the spleen of white goose (the Chinese goose, Anser cygnoides). The cDNA of goose IFNAR1 consisted of 1616 bp and encoded 406 amino acids with a predicted molecular weight of 46.4 kDa, while the cDNA of goose IFNAR2 consisted of 1525 bp and encoded 294 amino acids with a predicted molecular weight of 32.6 kDa. The IFNAR1 shared 85.4% identity in deduced amino acid sequence with duck IFNAR1, while IFNAR2 amino acid sequence showed 86% identity with that of duck IFNAR2. The age-related analysis of gene expression revealed that goose IFNα and IFNARs were all highly transcribed in pancreas, which may due to a reasonable amount of dendritic cells aggregated in pancreas. And goose IFNα and its cognate receptors had different structural features and tissue expression patterns during the period from embryonic goose to adult goose, suggesting that IFNα and IFNARs may maintain a developmental dynamic immune competence in unstimulated states. The data provided in this study may contribute to future understanding of the interaction between interferon and interferon receptors in immune mechanism. And it also helps us to understand the age-related susceptibility to pathogens in birds better.

  9. Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

    PubMed

    Fregien, N; Dolecki, G J; Mandel, M; Humphreys, T

    1983-06-01

    Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA. PMID:6688291

  10. [Molecular cloning, tissue distribution and expression in engineered cells of human orphan receptor GPR81].

    PubMed

    Wu, Fang-Ming; Huang, Huo-Gao; Hu, Ming; Gao, Yue; Liu, Yong-Xue

    2006-05-01

    The gpr81 was amplified by polymerase chain reaction (PCR) using human fetus kidney cDNA and whole blood genome DNA as template, respectively. The expression profile of gpr81 in human fetus was analyzed by RT-PCR and the result indicated GPR81 mRNA was most abundant in fetus liver and heart. In addition, the deduced amino acid of GPR81 was compared with other related molecules by Clustal w/x software, and a molecular phylogenetic tree was constructed with Treeview software. It was showed that GPR81 had the highest homology with nicotinic acid receptor in amino acids. After sequence identification, gpr81 was inserted into the plasmid pcDNA3. 1 (-)/his-mycA and then transfected into Chinese hamster ovary cell (CHO-K1). With the selection of G418, an engineered cell line which could stably express gpr81 was obtained by the indication of RT-PCR and Western-blot detection. The establishment of the cell line will serve as means for further study of GPR81.

  11. Early events in tissues during infection with pathogenic (SIVmac239) and nonpathogenic (SIVmac1A11) molecular clones of simian immunodeficiency virus.

    PubMed Central

    Lackner, A. A.; Vogel, P.; Ramos, R. A.; Kluge, J. D.; Marthas, M.

    1994-01-01

    The extent of virus replication, tissue distribution, localization of virus within tissues, and the presence of pathological lesions was examined early after experimental infection of rhesus monkeys with simian immunodeficiency virus (SIV). Three strains of SIV were used: molecularly cloned pathogenic SIVmac239; molecularly cloned nonpathogenic SIVmac1A11; and uncloned pathogenic SIVmac. The major targets of infection in all animals at 2 weeks postinoculation were the thymus and spleen. The distribution of virus within lymphoid organs varied with the viral inoculum: nonpathogenic SIVmac1A11 was present primarily within lymphoid follicles and in the thymic cortex; SIVmac239 was present primarily within periarteriolar lymphoid sheaths in the spleen, the paracortex of lymph nodes, and the medulla of the thymus; uncloned SIVmac was present in all these areas but tended to parallel the distribution of SIVmac239. Animals inoculated with nonpathogenic SIVmac1A11 had fewer SIV-positive cells by in situ hybridization and after 13 weeks postinoculation, virus was undetectable in any tissue from these animals. No significant pathological abnormalities were recognized in animals inoculated with this nonpathogenic virus. In contrast, nearly half of the animals inoculated with either SIVmac or SIVmac239 developed significant pathological lesions, including opportunistic infections by 13 weeks postinoculation, highlighting the virulence of these viruses. Our results indicate marked differences in tissue distribution between pathogenic and nonpathogenic molecular clones of SIV during the acute phase of infection. The most striking differences were the absence of SIVmac1A11 from the central nervous system and thymic medulla. The prominent early involvement of the thymus suggests that infection of this organ is a key event in the induction of immune suppression by SIV. Images Figure 1 Figure 2 Figure 3 PMID:8053500

  12. Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin.

    PubMed

    Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

    2016-01-01

    P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

  13. Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin

    PubMed Central

    Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

    2016-01-01

    P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

  14. Allograft inflammatory factor-1 in disk abalone (Haliotis discus discus): molecular cloning, transcriptional regulation against immune challenge and tissue injury.

    PubMed

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Kim, Yucheol; Oh, Chulhong; Kang, Do-Hyung; Whang, Ilson; Kim, Se-Jae; Lee, Jae-Seong; Choi, Cheol Young; Lee, Jehee

    2010-08-01

    Here, we report the identification and characterization of allograft inflammatory factor-1 (AIF-1) from disk abalone Haliotis discus discus that was denoted as AbAIF-1. The full-length cDNA of AbAIF-1 consists of a coding region (453 bp) for 151 amino acids with a 17 kDa molecular mass. Analysis of AbAIF-1 sequence showed that it shares characteristic two EF hand Ca(+2)-binding motifs. Results from phylogenetic analysis further confirm that AbAIF-1 is a member of the AIF-1 family similar to invertebrate and vertebrate counterparts suggesting it has high evolutional conservation. Tissue-specific expression and transcriptional regulation of AbAIF-1 were analyzed after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes), viral hemorrhagic septicemia virus (VHSV) immune challenge and during tissue injury by quantitative real-time PCR. It is shown that the expression of AbAIF-1 mRNA was expressed ubiquitously in all selected tissues in constitutive manner showing the highest level in hemocytes. Upon bacteria and VHSV challenge, AbAIF-1 showed the significant up-regulation in hemocytes than gills. After the tissue injury in shell and mantle, AbAIF-1 and antioxidant selenium-dependant glutathione peroxidase (SeGPx) transcripts were significantly upregulated in abalone hemocytes. Taken together, these findings suggest that AIF-1 could response against the pathogenic challenge or tissue injury in abalone like mollusks. Also, AbAIF-1 may involve in wound healing and shell repair after the tissue injury of abalone.

  15. Molecular cloning, tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding

    NASA Astrophysics Data System (ADS)

    Xue, Chunyu; Xi, Bingwen; Ren, Mingchun; Dong, Jingjing; Xie, Jun; Xu, Pao

    2015-03-01

    Mucins are important components of mucus, which form a natural, physical, biochemical and semipermeable mucosal layer on the epidermis of fish gills, skin, and the gastrointestinal tract. As the first step towards characterizing the function of Muc2, we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp, and analyzed its tissue-specific expression pattern by quantitative real-time PCR (qPCR). The obtained sequence comprised 41 bp 5'-untranslated region (5'-UTR), 2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains (VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes ( β- Actin, RPI13α, RPII, 18S) for the qPCR, RPII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine, in the order (highest to lowest) middle-intestine > fore-intestine > hind-intestine. Muc2 was expressed relatively poorly in other organs (brain, liver, kidney, spleen, skin and gill). Furthermore, after 20-days of starvation, M. amblycephala Muc2 expressions after refeeding for 0 h, 3 h, 16 h, 3 d, and 10 d were significantly decreased in the three intestinal segments ( P<0.05) at 16 h, and were then upregulated to near the initial level at 10 d.

  16. Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning, tissue expression, and the function of feeding regulation.

    PubMed

    Wang, Tao; Zhou, Chaowei; Yuan, Dengyue; Lin, Fangjun; Chen, Hu; Wu, Hongwei; Wei, Rongbin; Xin, Zhiming; Liu, Ju; Gao, Yundi; Li, Zhiqiong

    2014-10-01

    Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.

  17. Molecular cloning of the human Hand1 gene/cDNA and its tissue-restricted expression in cytotrophoblastic cells and heart.

    PubMed

    Knöfler, M; Meinhardt, G; Vasicek, R; Husslein, P; Egarter, C

    1998-12-11

    The basic helix-loop-helix (bHLH) factor Hand1 plays a role in the developing chicken heart and is required for trophoblast giant cell differentiation and cardiac looping of mouse embryonic development. Here, we report the cloning of the human Hand1 cDNA and gene from a heart-specific cDNA library and a genomic lambda-DNA library, respectively. We present the nucleotide sequence of a 1.75kb cDNA clone, encoding the presumptive 215 amino acid human Hand1 protein, and show homology comparison of the conserved bHLH region between different species. In vitro transcription-translation of Hand1 mRNA and analysis of protein size suggest that the Hand1 polypeptide is (post)translationally modified. By Southern blot analysis we demonstrate that the isolated genomic DNA clone harbours the entire Hand1 gene and describe molecular structure and sequences of the two 799 and 938bp exons and the single 1.56kb intron. The expression pattern of the mRNA in different human tissues revealed that Hand1 transcripts are restricted to the heart, suggesting that the protein could be required for cardiac-specific gene transcription and function in adults. Hand1 transcripts were undetectable in a non-tumorigenic villous trophoblast cell line, immunopurified cytotrophoblasts undergoing in vitro differentiation, and first trimester placental tissue, suggesting that the transcription factor is not involved in the development of villous and extravillous trophoblast cell lineages. Hand1 mRNA, however, was abundantly expressed in cytotrophoblastic Jeg-3 and BeWo cells, suggesting that Hand1 could be required for early trophoblast differentiation.

  18. Molecular cloning and tissue-specific expression of a gonadotropin-releasing hormone receptor in the Japanese eel.

    PubMed

    Okubo, K; Suetake, H; Usami, T; Aida, K

    2000-08-01

    Gonadotropin-releasing hormone (GnRH) is a key regulatory neuropeptide involved in the control of reproduction in vertebrates. In the Japanese eel, one of the most primitive teleost species, two molecular forms of GnRH, mammalian-type GnRH and chicken-II-type GnRH (cGnRH-II), have been identified. This study has isolated a full-length cDNA for a GnRH receptor from the pituitary of the eel. The 3233-bp cDNA encodes a 380-amino acid protein which contains seven hydrophobic transmembrane domains and N- and C-terminal regions. The exon/intron organization of the open reading frame of the eel GnRH receptor gene was also determined. The open reading frame consists of three exons and two introns. The exon-intron splice site is similar to that of the GnRH receptor genes of mammals reported so far. Expression of the eel GnRH receptor was detected in various parts of the brain, pituitary, eye, olfactory epithelium, and testis. This result suggests that GnRH has local functions in these tissues in addition to its actions on gonadotropin synthesis and release in the pituitary. This tissue-specific expression pattern is similar to that of the eel cGnRH-II. Furthermore, the present eel receptor shows very high amino acid identity with the catfish and goldfish GnRH receptors, which are highly selective for the cGnRH-II. These results suggest that the cGnRH-II acts through binding to the present receptor in the eel.

  19. Molecular cloning, genomic organization, chromosome mapping, tissues expression pattern and identification of a novel splicing variant of porcine CIDEb gene.

    PubMed

    Li, YanHua; Li, AiHua; Yang, Z Q

    2016-09-01

    Cell death-inducing DNA fragmentation factor-α-like effector b (CIDEb) is a member of the CIDE family of apoptosis-inducing factors, CIDEa and CIDEc have been reported to be Lipid droplets (LDs)-associated proteins that promote atypical LD fusion in adipocytes, and responsible for liver steatosis under fasting and obese conditions, whereas CIDEb promotes lipid storage under normal diet conditions [1], and promotes the formation of triacylglyceride-enriched VLDL particles in hepatocytes [2]. Here, we report the gene cloning, chromosome mapping, tissue distribution, genetic expression analysis, and identification of a novel splicing variant of the porcine CIDEb gene. Sequence analysis shows that the open reading frame of the normal porcine CIDEb isoform covers 660bp and encodes a 219-amino acid polypeptide, whereas its alternative splicing variant encodes a 142-amino acid polypeptide truncated at the fourth exon and comprised of the CIDE-N domain and part of the CIDE-C domain. The deduced amino acid sequence of normal porcine CIDEb shows an 85.8% similarity to the human protein and 80.0% to the mouse protein. The CIDEb genomic sequence spans approximately 6KB comprised of five exons and four introns. Radiation hybrid mapping demonstrated that porcine CIDEb is located at chromosome 7q21 and at a distance of 57cR from the most significantly linked marker, S0334, regions that are syntenic with the corresponding region in the human genome. Tissue expression analysis indicated that normal CIDEb mRNA is ubiquitously expressed in many porcine tissues. It was highly expressed in white adipose tissue and was observed at relatively high levels in the liver, lung, small intestine, lymphatic tissue and brain. The normal version of CIDEb was the predominant form in all tested tissues, whereas the splicing variant was expressed at low levels in all examined tissues except the lymphatic tissue. Furthermore, genetic expression analysis indicated that CIDEb mRNA levels were

  20. Molecular cloning, genomic organization, chromosome mapping, tissues expression pattern and identification of a novel splicing variant of porcine CIDEb gene.

    PubMed

    Li, YanHua; Li, AiHua; Yang, Z Q

    2016-09-01

    Cell death-inducing DNA fragmentation factor-α-like effector b (CIDEb) is a member of the CIDE family of apoptosis-inducing factors, CIDEa and CIDEc have been reported to be Lipid droplets (LDs)-associated proteins that promote atypical LD fusion in adipocytes, and responsible for liver steatosis under fasting and obese conditions, whereas CIDEb promotes lipid storage under normal diet conditions [1], and promotes the formation of triacylglyceride-enriched VLDL particles in hepatocytes [2]. Here, we report the gene cloning, chromosome mapping, tissue distribution, genetic expression analysis, and identification of a novel splicing variant of the porcine CIDEb gene. Sequence analysis shows that the open reading frame of the normal porcine CIDEb isoform covers 660bp and encodes a 219-amino acid polypeptide, whereas its alternative splicing variant encodes a 142-amino acid polypeptide truncated at the fourth exon and comprised of the CIDE-N domain and part of the CIDE-C domain. The deduced amino acid sequence of normal porcine CIDEb shows an 85.8% similarity to the human protein and 80.0% to the mouse protein. The CIDEb genomic sequence spans approximately 6KB comprised of five exons and four introns. Radiation hybrid mapping demonstrated that porcine CIDEb is located at chromosome 7q21 and at a distance of 57cR from the most significantly linked marker, S0334, regions that are syntenic with the corresponding region in the human genome. Tissue expression analysis indicated that normal CIDEb mRNA is ubiquitously expressed in many porcine tissues. It was highly expressed in white adipose tissue and was observed at relatively high levels in the liver, lung, small intestine, lymphatic tissue and brain. The normal version of CIDEb was the predominant form in all tested tissues, whereas the splicing variant was expressed at low levels in all examined tissues except the lymphatic tissue. Furthermore, genetic expression analysis indicated that CIDEb mRNA levels were

  1. Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression.

    PubMed

    Yan, Aifen; Zhang, Lingjuang; Tang, Zhiguo; Zhang, Yanhong; Qin, Chaobin; Li, Bo; Li, Wensheng; Lin, Haoran

    2011-07-01

    Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro.

  2. Molecular characterization of the gonadal kisspeptin system: Cloning, tissue distribution, gene expression analysis and localization in sablefish (Anoplopoma fimbria).

    PubMed

    Fairgrieve, Marian R; Shibata, Yasushi; Smith, Elizabeth K; Hayman, Edward S; Luckenbach, J Adam

    2016-01-01

    The kisspeptin system plays pivotal roles in the regulation of vertebrate reproduction. Classically, kisspeptin produced in the brain stimulates brain gonadotropin-releasing hormone signaling, which in turn activates the pituitary-gonad axis. Expression of the kisspeptin system has also been documented in peripheral tissues, including gonads of mammals and fishes. However, the fish gonadal kisspeptin system remained uncharacterized. Herein we report identification and characterization of four kisspeptin system mRNAs (kisspeptin 1 (kiss1), kiss2, and G protein-coupled receptor 54-1 (gpr54-1) and gpr54-2) in sablefish, Anoplopoma fimbria. Sablefish predicted protein sequences were highly similar to those of other marine teleosts, but less so to freshwater teleosts. Tissue distribution analyses revealed that all four kisspeptin-system transcripts were expressed in both brain and gonad. However, kiss2 was the predominant transcript in the gonads and the only transcript detected in ovulated eggs. Ontogenetic analysis of kiss2 expression in juvenile sablefish gonads demonstrated that levels were low during sex differentiation but increased with fish size and gonadal development. Dramatic increases in kiss2 mRNA occurred during primary oocyte growth, while levels remained relatively low in testes. In situ hybridization revealed that kiss2 mRNA was localized to cytoplasm of perinucleolus stage oocytes, suggesting it could play a local role in oogenesis or could be synthesized and stored within oocytes as maternal mRNA. This represents the first study to focus on the gonadal kisspeptin system in fishes and provides important tools for further investigation of both the gonadal and brain kisspeptin systems in sablefish.

  3. Molecular characterization of the gonadal kisspeptin system: Cloning, tissue distribution, gene expression analysis and localization in sablefish (Anoplopoma fimbria).

    PubMed

    Fairgrieve, Marian R; Shibata, Yasushi; Smith, Elizabeth K; Hayman, Edward S; Luckenbach, J Adam

    2016-01-01

    The kisspeptin system plays pivotal roles in the regulation of vertebrate reproduction. Classically, kisspeptin produced in the brain stimulates brain gonadotropin-releasing hormone signaling, which in turn activates the pituitary-gonad axis. Expression of the kisspeptin system has also been documented in peripheral tissues, including gonads of mammals and fishes. However, the fish gonadal kisspeptin system remained uncharacterized. Herein we report identification and characterization of four kisspeptin system mRNAs (kisspeptin 1 (kiss1), kiss2, and G protein-coupled receptor 54-1 (gpr54-1) and gpr54-2) in sablefish, Anoplopoma fimbria. Sablefish predicted protein sequences were highly similar to those of other marine teleosts, but less so to freshwater teleosts. Tissue distribution analyses revealed that all four kisspeptin-system transcripts were expressed in both brain and gonad. However, kiss2 was the predominant transcript in the gonads and the only transcript detected in ovulated eggs. Ontogenetic analysis of kiss2 expression in juvenile sablefish gonads demonstrated that levels were low during sex differentiation but increased with fish size and gonadal development. Dramatic increases in kiss2 mRNA occurred during primary oocyte growth, while levels remained relatively low in testes. In situ hybridization revealed that kiss2 mRNA was localized to cytoplasm of perinucleolus stage oocytes, suggesting it could play a local role in oogenesis or could be synthesized and stored within oocytes as maternal mRNA. This represents the first study to focus on the gonadal kisspeptin system in fishes and provides important tools for further investigation of both the gonadal and brain kisspeptin systems in sablefish. PMID:26386183

  4. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.

    PubMed

    Langbein, L; Heid, H W; Moll, I; Franke, W W

    1993-12-01

    Differentiation of human plantar and palmar epidermis is characterized by the suprabasal synthesis of a major special intermediate-sized filament (IF) protein, the type I (acidic) cytokeratin 9 (CK 9). Using partial amino acid (aa) sequence information obtained by direct Edman sequencing of peptides resulting from proteolytic digestion of purified CK 9, we synthesized several redundant primers by 'back-translation'. Amplification by polymerase chain reaction (PCR) of cDNAs obtained by reverse transcription of mRNAs from human foot sole epidermis, including 5'-primer extension, resulted in multiple overlapping cDNA clones, from which the complete cDNA (2353 bp) could be constructed. This cDNA encoded the CK 9 polypeptide with a calculated molecular weight of 61,987 and an isoelectric point at about pH 5.0. The aa sequence deduced from cDNA was verified in several parts by comparison with the peptide sequences and showed the typical structure of type I CKs, with a head (153 aa), and alpha-helical coiled-coil-forming rod (306 aa), and a tail (163 aa) domain. The protein displayed the highest homology to human CK 10, not only in the highly conserved rod domain but also in large parts of the head and the tail domains. On the other hand, the aa sequence revealed some remarkable differences from CK 10 and other CKs, even in the most conserved segments of the rod domain. The nuclease digestion pattern seen on Southern blot analysis of human genomic DNA indicated the existence of a unique CK 9 gene. Using CK 9-specific riboprobes for hybridization on Northern blots of RNAs from various epithelia, a mRNA of about 2.4 kb in length could be identified only in foot sole epidermis, and a weaker cross-hybridization signal was seen in RNA from bovine heel pad epidermis at about 2.0 kb. A large number of tissues and cell cultures were examined by PCR of mRNA-derived cDNAs, using CK 9-specific primers. But even with this very sensitive signal amplification, only palmar

  5. Molecular cloning and tissue distribution of peroxisome proliferator-activated receptor-alpha (PPARα) and gamma (PPARγ) in the pigeon (Columba livia domestica).

    PubMed

    Xie, P; Yuan, C; Wang, C; Zou, X-T; Po, Z; Tong, H-B; Zou, J-M

    2014-01-01

    1. Peroxisome proliferator-activated receptors (PPAR) are involved in lipid metabolism through transcriptional regulation of target gene expression. The objective of the current study was to clone and characterise the PPARα and PPARγ genes in pigeon. 2. The full-length of 1941-bp PPARα and 1653-bp PPARγ were cloned from pigeons. The two genes were predicted to encode 468 and 475 amino acids, respectively. Both proteins contained two C4-type zinc fingers, a nuclear hormone receptor DNA-binding region signature and a HOLI domain (ligand binding domain of hormone receptors), and had high identities with other corresponding avian genes. 3. Using quantitative real-time PCR, pigeon PPARα gene expression was shown to be high in kidney, liver, gizzard and duodenum whereas PPARγ was predominantly expressed in adipose tissue.

  6. Molecular cloning and tissue distribution of cholecystokinin-1 receptor (CCK-1R) in yellowtail Seriola quinqueradiata and its response to feeding and in vitro CCK treatment.

    PubMed

    Furutani, Takahiro; Masumoto, Toshiro; Fukada, Haruhisa

    2013-06-01

    In vertebrates, the peptide cholecystokinin (CCK) is one of the most important neuroregulatory digestive hormones. CCK acts via CCK receptors that are classified into two subtypes, CCK-1 receptor (CCK-1R; formally CCK-A) and CCK-2 receptor (formally CCK-B). In particular, the CCK-1R is involved in digestion and is regulated by CCK. However, very little information is known about CCK-1R in fish. Therefore, we performed molecular cloning of CCK-1R cDNA from the digestive tract of yellowtail Seriola quinqueradiata. Phylogenetic tree analysis showed a high sequence identity between the cloned yellowtail CCK receptor cDNA and CCK-1R, which belongs to the CCK-1R cluster. Furthermore, the expression of yellowtail CCK receptor mRNA was observed in gallbladder, pyloric caeca, and intestines, similarly to CCK-1R mRNA expression in mammals, suggesting that the cloned cDNA is of CCK-1R from yellowtail. In in vivo experiments, the CCK-1R mRNA levels increased in the gallbladder and pyloric caeca after feeding, whereas in vitro, mRNA levels of CCK-1R and digestive enzymes in cultured pyloric caeca increased by the addition of CCK. These results suggest that CCK-1R plays an important role in digestion stimulated by CCK in yellowtail. PMID:23467070

  7. Molecular cloning, characterization, tissue distribution and mRNA expression changes during the hibernation and reproductive periods of estrogen receptor alpha (ESR1) in Chinese alligator, Alligator sinensis.

    PubMed

    Zhang, Ruidong; Hu, Yuehong; Wang, Huan; Yan, Peng; Zhou, Yongkang; Wu, Rong; Wu, Xiaobing

    2016-10-01

    Chinese alligator, Alligator sinensis, is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha (ERα/ESR1) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period (P<0.05) in cerebellum, pituitary gland, liver, spleen, lung, kidney and ovary, while no significant change in other examined tissues (P>0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles. PMID:27212643

  8. Porcine MAP3K5 analysis: molecular cloning, characterization, tissue expression pattern, and copy number variations associated with residual feed intake.

    PubMed

    Pu, L; Zhang, L C; Zhang, J S; Song, X; Wang, L G; Liang, J; Zhang, Y B; Liu, X; Yan, H; Zhang, T; Yue, J W; Li, N; Wu, Q Q; Wang, L X

    2016-01-01

    Mitogen-activated protein kinase kinase kinase 5 (MAP3K5) is essential for apoptosis, proliferation, differentiation, and immune responses, and is a candidate marker for residual feed intake (RFI) in pig. We cloned the full-length cDNA sequence of porcine MAP3K5 by rapid-amplification of cDNA ends. The 5451-bp gene contains a 5'-untranslated region (UTR) (718 bp), a coding region (3738 bp), and a 3'-UTR (995 bp), and encodes a peptide of 1245 amino acids, which shares 97, 99, 97, 93, 91, and 84% sequence identity with cattle, sheep, human, mouse, chicken, and zebrafish MAP3K5, respectively. The deduced MAP3K5 protein sequence contains two conserved domains: a DUF4071 domain and a protein kinase domain. Phylogenetic analysis showed that porcine MAP3K5 forms a separate branch to vicugna and camel MAP3K5. Tissue expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR) revealed that MAP3K5 was expressed in the heart, liver, spleen, lung, kidney, muscle, fat, pancrea, ileum, and stomach tissues. Copy number variation was detected for porcine MAP3K5 and validated by qRT-PCR. Furthermore, a significant increase in average copy number was detected in the low RFI group when compared to the high RFI group in a Duroc pig population. These results provide useful information regarding the influence of MAP3K5 on RFI in pigs. PMID:27525933

  9. Leptin and cholecystokinin in Schizothorax prenanti: molecular cloning, tissue expression, and mRNA expression responses to periprandial changes and fasting.

    PubMed

    Yuan, Dengyue; Wang, Tao; Zhou, Chaowei; Lin, Fangjun; Chen, Hu; Wu, Hongwei; Wei, Rongbin; Xin, Zhiming; Li, Zhiqiong

    2014-08-01

    In the present study, full-length cDNA sequences of leptin and cholecystokinin (CCK) were cloned from Schizothorax prenanti (S. prenanti), and applied real-time quantitative PCR to characterize the tissue distribution, and appetite regulatory effects of leptin and CCK in S. prenanti. The S. prenanti leptin and CCK full-length cDNA sequences were 1121 bp and 776 bp in length, encoding the peptide of 171 and 123 amino acid residues, respectively. Tissue distribution analysis showed that leptin mRNA was mainly expressed in the liver of S. prenanti. CCK was widely expressed, with the highest levels of expression in the hypothalamus, myelencephalon, telencephalon and foregut of S. prenanti. The CCK mRNA expression was highly elevated after feeding, whereas the leptin mRNA expression was not affected by single meal. These results suggested that CCK is a postprandial satiety signal in S. prenanti, but leptin might not be. In present study, leptin and CCK gene expression were both decreased after fasting and increased after refeeding, which suggested leptin and CCK might be involved in regulation of appetite in S. prenanti. This study provides an essential groundwork to further elucidate the appetite regulatory systems of leptin and CCK in S. prenanti as well as in other teleosts.

  10. Molecular cloning and in silico analysis of the duck (Anas platyrhynchos) MEF2A gene cDNA and its expression profile in muscle tissues during fetal development

    PubMed Central

    Liu, Hehe; Wang, Jiwen; Si, Jianmin; Jia, Jing; Li, Liang; Han, Chunchun; Huang, Kailiang; He, Hua; Xu, Feng

    2012-01-01

    The role of myogenic enhancer transcription factor 2a (MEF2A) in avian muscle during fetal development is unknown. In this work, we cloned the duck MEF2A cDNA sequence (GenBank accession no. HM460752) and examined its developmental expression profiles in cardiac muscle, non-vascular smooth muscle and skeletal muscle. Duck MEF2A cDNA comprised 1479 bp encoding 492 amino acid residues. In silico analysis showed that MEF2A contained MADS (MCM1, AGAMOUS, DEFICIENS and SRF - serum response factor), MEF2 and mitogen-activated protein kinase (MAPK) transcription domains with high homology to related proteins in other species. Modified sites in these domains were conserved among species and several variants were found. Quantitative PCR showed that MEF2A was expressed in all three muscles at each developmental stage examined, with the expression in smooth muscle being higher than in the other muscles. These results indicate that the conserved domains of duck MEF2A, including the MADS and MEF2 domains, are important for MEF2A transcription factor function. The expression of MEF2A in duck smooth muscle and cardiac muscle suggests that MEF2A plays a role in these two tissues. PMID:22481893

  11. Molecular cloning and characterization of the human folate-binding protein cDNA from placenta and malignant tissue culture (KB) cells.

    PubMed

    Elwood, P C

    1989-09-01

    Human folate-binding proteins (FBPs) are single chain glycoproteins that contain a high affinity binding site for folates and methotrexate and occur in a soluble or membrane-associated form. The membrane-associated FBP is involved in the uptake of physiologic folates and methotrexate. In this study, human FBP cDNA clones were isolated from human malignant nasopharyngeal carcinoma (KB) cell and placental cDNA libraries by means of oligonucleotide probes derived from determined internal amino acid sequences. The longest cDNA nucleotide sequence is 1126 base pairs and encodes a polypeptide that contains 257 amino acid residues (calculated molecular mass = 29,817). The deduced amino acid sequence is 80% homologous to a bovine soluble FBP, is greater than 99% homologous to the reported partial amino acid sequence of the human soluble FBP, contains three potential N-linked glycosylation sites, and has hydrophobic amino- and carboxylterminal regions which are consistent with a signal peptide and a potential membrane-anchoring domain, respectively. On Northern blot analysis, radiolabeled cDNA probes hybridize to a single 1100-base pair mRNA species that is expressed to a variable degree in human KB cells, placenta, brain, and epithelial mRNA but is not detectable in human liver mRNA. In vitro translation of RNA transcripts from the FBP cDNA inserts yields a 30-kDa and a 42-kDa polypeptide in the absence and presence of microsomal membranes, respectively. PMID:2768245

  12. Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors.

    PubMed

    Tian, Xue; Meng, Xiaolin; Wang, Liangyan; Song, Yunfei; Zhang, Danli; Ji, Yuankai; Li, Xuejun; Dong, Changsheng

    2015-01-25

    Slc7a11 encoding solute carrier family 7 member 11 (amionic amino acid transporter light chain, xCT), has been identified to be a critical genetic regulator of pheomelanin synthesis in hair and melanocytes. To better understand the molecular characterization of Slc7a11 and the expression patterns in skin of white versus brown alpaca (lama paco), we cloned the full length coding sequence (CDS) of alpaca Slc7a11 gene and analyzed the expression patterns using Real Time PCR, Western blotting and immunohistochemistry. The full length CDS of 1512bp encodes a 503 amino acid polypeptide. Sequence analysis showed that alpaca xCT contains 12 transmembrane regions consistent with the highly conserved amino acid permease (AA_permease_2) domain similar to other vertebrates. Sequence alignment and phylogenetic analysis revealed that alpaca xCT had the highest identity and shared the same branch with Camelus ferus. Real Time PCR and Western blotting suggested that xCT was expressed at significantly high levels in brown alpaca skin, and transcripts and protein possessed the same expression pattern in white and brown alpaca skins. Additionally, immunohistochemical analysis further demonstrated that xCT staining was robustly increased in the matrix and root sheath of brown alpaca skin compared with that of white. These results suggest that Slc7a11 functions in alpaca coat color regulation and offer essential information for further exploration on the role of Slc7a11 in melanogenesis. PMID:25455099

  13. Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors.

    PubMed

    Tian, Xue; Meng, Xiaolin; Wang, Liangyan; Song, Yunfei; Zhang, Danli; Ji, Yuankai; Li, Xuejun; Dong, Changsheng

    2015-01-25

    Slc7a11 encoding solute carrier family 7 member 11 (amionic amino acid transporter light chain, xCT), has been identified to be a critical genetic regulator of pheomelanin synthesis in hair and melanocytes. To better understand the molecular characterization of Slc7a11 and the expression patterns in skin of white versus brown alpaca (lama paco), we cloned the full length coding sequence (CDS) of alpaca Slc7a11 gene and analyzed the expression patterns using Real Time PCR, Western blotting and immunohistochemistry. The full length CDS of 1512bp encodes a 503 amino acid polypeptide. Sequence analysis showed that alpaca xCT contains 12 transmembrane regions consistent with the highly conserved amino acid permease (AA_permease_2) domain similar to other vertebrates. Sequence alignment and phylogenetic analysis revealed that alpaca xCT had the highest identity and shared the same branch with Camelus ferus. Real Time PCR and Western blotting suggested that xCT was expressed at significantly high levels in brown alpaca skin, and transcripts and protein possessed the same expression pattern in white and brown alpaca skins. Additionally, immunohistochemical analysis further demonstrated that xCT staining was robustly increased in the matrix and root sheath of brown alpaca skin compared with that of white. These results suggest that Slc7a11 functions in alpaca coat color regulation and offer essential information for further exploration on the role of Slc7a11 in melanogenesis.

  14. Cholesterol biosynthesis from lanosterol. Molecular cloning, tissue distribution, expression, chromosomal localization, and regulation of rat 7-dehydrocholesterol reductase, a Smith-Lemli-Opitz syndrome-related protein.

    PubMed

    Bae, S H; Lee, J N; Fitzky, B U; Seong, J; Paik, Y K

    1999-05-21

    The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and developmental abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization. Rat DHCR, calculated molecular mass of 54.15-kDa polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 and 87%, respectively) as compared with its other related proteins (e.g. fungal sterol Delta14-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains. Five putative sterol-sensing domains were predicted to be localized in transmembrane domains 4-8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein. The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysis indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain. In rat brains, the highest level of mRNA encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla. Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increase of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant reduction in serum total cholesterol level were observed. However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at

  15. Cloning

    MedlinePlus

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  16. Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification.

    PubMed Central

    Pääbo, S

    1989-01-01

    Several chemical and enzymatic properties were examined in the DNA extracted from dry remains of soft tissues that vary in age from 4 to 13,000 years and represent four species, including two extinct animals (the marsupial wolf and giant ground sloth). The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, which primarily manifest themselves as modifications of pyrimidines and sugar residues as well as baseless sites and intermolecular cross-links. This renders molecular cloning difficult. However, the polymerase chain reaction can be used to amplify and study short mitochondrial DNA sequences that are of anthropological and evolutionary significance. This opens up the prospect of performing diachronical studies of molecular evolutionary genetics. Images PMID:2928314

  17. Molecular cloning and tissue-specific transcriptional regulation of the first peroxidase family member, Udp1, in stinging nettle (Urtica dioica).

    PubMed

    Douroupi, Triantafyllia G; Papassideri, Issidora S; Stravopodis, Dimitrios J; Margaritis, Lukas H

    2005-12-01

    A full-length cDNA clone, designated Udp1, was isolated from Urtica dioica (stinging nettle), using a polymerase chain reaction based strategy. The putative Udp1 protein is characterized by a cleavable N-terminal signal sequence, likely responsible for the rough endoplasmic reticulum entry and a 310 amino acids mature protein, containing all the important residues, which are evolutionary conserved among different members of the plant peroxidase family. A unique structural feature of the Udp1 peroxidase is defined into the short carboxyl-terminal extension, which could be associated with the vacuolar targeting process. Udp1 peroxidase is differentially regulated at the transcriptional level and is specifically expressed in the roots. Interestingly, wounding and ultraviolet radiation stress cause an ectopic induction of the Udp1 gene expression in the aerial parts of the plant. A genomic DNA fragment encoding the Udp1 peroxidase was also cloned and fully sequenced, revealing a structural organization of three exons and two introns. The phylogenetic relationships of the Udp1 protein to the Arabidopsis thaliana peroxidase family members were also examined and, in combination with the homology modelling approach, dictated the presence of distinct structural elements, which could be specifically involved in the determination of substrate recognition and subcellular localization of the Udp1 peroxidase.

  18. Molecular cloning and tissue-specific transcriptional regulation of the first peroxidase family member, Udp1, in stinging nettle (Urtica dioica).

    PubMed

    Douroupi, Triantafyllia G; Papassideri, Issidora S; Stravopodis, Dimitrios J; Margaritis, Lukas H

    2005-12-01

    A full-length cDNA clone, designated Udp1, was isolated from Urtica dioica (stinging nettle), using a polymerase chain reaction based strategy. The putative Udp1 protein is characterized by a cleavable N-terminal signal sequence, likely responsible for the rough endoplasmic reticulum entry and a 310 amino acids mature protein, containing all the important residues, which are evolutionary conserved among different members of the plant peroxidase family. A unique structural feature of the Udp1 peroxidase is defined into the short carboxyl-terminal extension, which could be associated with the vacuolar targeting process. Udp1 peroxidase is differentially regulated at the transcriptional level and is specifically expressed in the roots. Interestingly, wounding and ultraviolet radiation stress cause an ectopic induction of the Udp1 gene expression in the aerial parts of the plant. A genomic DNA fragment encoding the Udp1 peroxidase was also cloned and fully sequenced, revealing a structural organization of three exons and two introns. The phylogenetic relationships of the Udp1 protein to the Arabidopsis thaliana peroxidase family members were also examined and, in combination with the homology modelling approach, dictated the presence of distinct structural elements, which could be specifically involved in the determination of substrate recognition and subcellular localization of the Udp1 peroxidase. PMID:16219430

  19. Molecular cloning, sequencing and tissue expression of vasotocin and isotocin precursor genes from Ostariophysian catfishes: phylogeny and evolutionary considerations in teleosts

    PubMed Central

    Banerjee, Putul; Chaube, Radha; Joy, Keerikkattil P.

    2015-01-01

    Basic and neutral neurohypophyseal (NH) nonapeptides have evolved from vasotocin (VT) by a gene duplication at the base of the gnathostome lineage. In teleosts, VT and IT are the basic and neutral peptides, respectively. In the present study, VT and IT precursor genes of Heteropneustes fossilis and Clarias batrachus (Siluriformes, Ostariophysi) were cloned and sequenced. The channel catfish Icatalurus punctatus NH precursor sequences were obtained from EST database. The catfish NH sequences were used along with the available Acanthopterygii and other vertebrate NH precursor sequences to draw phylogenetic inference on the evolutionary history of the teleost NH peptides. Synteny analysis of the NH gene loci in various teleost species was done to complement the phylogenetic analysis. In H. fossilis, the NH transcripts were also sequenced from the ovary. The cloned genes and the deduced precursor proteins showed conserved characteristics of the NH nonapeptide precursors. The genes are expressed in brain and ovary (follicular envelope) of H. fossilis with higher transcript abundance in the brain. The addition of the catfish sequences in the phylogenetic analysis revealed that the VT and IT precursors of the species-rich superorders of teleosts have a distinct phylogenetic history with the Acanthopterygii VT and IT precursors sharing a less evolutionary distance and the Ostariophysi VT and IT having a greater evolutionary distance. The genomic location of VT and IT precursors, and synteny analysis of the NH loci lend support to the phylogenetic inference and suggest a footprint of fish- specific whole genome duplication (3R) and subsequent diploidization in the NH loci. The VT and IT precursor genes are most likely lineage-specific paralogs resulting from differential losses of the 3R NH paralogs in the two superorders. The independent yet consistent retention of VT and IT in the two superorders might be directed by a stringent ligand-receptor selectivity. PMID:26029040

  20. Molecular cloning of protein-based polymers.

    PubMed

    Mi, Lixin

    2006-07-01

    Protein-based biopolymers have become a promising class of materials for both biomedical and pharmaceutical applications, as they have well-defined molecular weights, monomer compositions, as well as tunable chemical, biological, and mechanical properties. Using standard molecular biology tools, it is possible to design and construct genes encoding artificial proteins or protein-based polymers containing multiple repeats of amino acid sequences. This article reviews some of the traditional methods used for constructing DNA duplexes encoding these repeat-containing genes, including monomer generation, concatemerization, iterative oligomerization, and seamless cloning. A facile and versatile method, called modules of degenerate codons (MDC), which uses PCR and codon degeneracy to overcome some of the disadvantages of traditional methods, is introduced. Re-engineering of the random coil spacer domain of a bioactive protein, WPT2-3R, is used to demonstrate the utility of the MDC method. MDC re-constructed coding sequences facilitate further manipulations, such as insertion, deletion, and swapping of various sequence modules. A summary of some promising emerging techniques for synthesizing repetitive sequence-containing artificial proteins is also provided. PMID:16827576

  1. Should we clone human beings? Cloning as a source of tissue for transplantation.

    PubMed Central

    Savulescu, J

    1999-01-01

    The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus. PMID:10226910

  2. Should we clone human beings? Cloning as a source of tissue for transplantation.

    PubMed

    Savulescu, J

    1999-04-01

    The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus.

  3. Molecular cloning of a small DNA binding protein with specificity for a tissue-specific negative element within the rps1 promoter.

    PubMed Central

    Zhou, D X; Bisanz-Seyer, C; Mache, R

    1995-01-01

    A cDNA encoding a specific binding activity for the tissue-specific negative cis-element S1F binding site of spinach rps1 was isolated from a spinach cDNA expression library. This cDNA of 0.7 kb encodes an unusual small peptide of only 70 amino acids, with a basic domain which contains a nuclear localization signal and a putative DNA binding helix. This protein, named S1Fa, is highly conserved between dicotyledonous and monocotyledonous plants and may represent a novel class of DNA binding proteins. The corresponding mRNA is accumulated more in roots and in etiolated seedlings than in green leaves. This expression pattern is correlated with the tissue-specific function of the S1F binding site which represses the rps1 promoter preferentially in roots and in etiolated plants. Images PMID:7739894

  4. Insulin-like growth factor-binding protein-1 (IGFBP-1) in goldfish, Carassius auratus: molecular cloning, tissue expression, and mRNA expression responses to periprandial changes and cadmium exposure.

    PubMed

    Chen, Wenbo; Zhang, Zhen; Dong, Haiyan; Yan, Fangfang

    2016-06-01

    In this study, the cDNA encoding insulin-like growth factor-binding protein-1 (IGFBP-1) was cloned from the liver of goldfish (Carassius auratus). The obtained goldfish IGFBP-1 cDNA sequence was 1037 bp in length and had an open reading frame of 789 bp encoding a predicted polypeptide of 262 amino acid residues. IGFBP-1 transcript was detected in all tested central nervous and peripheral tissues. The relatively higher levels of IGFBP-1 mRNA were observed in the liver, gill, kidney, heart, spleen, fat and testis, while the lower levels were found in all different regions of brain, muscle and intestine. In the skin, IGFBP-1 mRNA expression level was extremely low. The IGFBP-1 mRNA expression level in liver was significantly elevated after feeding. With cadmium exposure for 24 h, IGFBP-1 mRNA expression levels in spleen and liver were significantly increased at different cadmium concentrations ranging from 0.5 to 10 ppm. The results in this study provided the data regarding molecular characteristics and expression patterns of IGFBP-1 in goldfish and showed that the expression of IGFBP-1 mRNA might be associated with metabolic status and heavy metal stress and regulated by metabolic factors and cadmium in fish. PMID:26753895

  5. Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue

    PubMed Central

    Kang, Jin-Ho; Campos, Marcelo L.; Zemelis-Durfee, Starla; Al-Haddad, Jameel M.; Jones, A. Daniel; Telewski, Frank W.; Brandizzi, Federica; Howe, Gregg A.

    2016-01-01

    Trichomes are epidermal structures that provide a first line of defense against arthropod herbivores. The recessive hairless (hl) mutation in tomato (Solanum lycopersicum L.) causes severe distortion of trichomes on all aerial tissues, impairs the accumulation of sesquiterpene and polyphenolic compounds in glandular trichomes, and compromises resistance to the specialist herbivore Manduca sexta. Here, we demonstrate that the tomato Hl gene encodes a subunit (SRA1) of the highly conserved WAVE regulatory complex that controls nucleation of actin filaments in a wide range of eukaryotic cells. The tomato SRA1 gene spans a 42-kb region containing both Solyc11g013280 and Solyc11g013290. The hl mutation corresponds to a complex 3-kb deletion that removes the last exon of the gene. Expression of a wild-type SRA1 cDNA in the hl mutant background restored normal trichome development, accumulation of glandular trichome-derived metabolites, and resistance to insect herbivory. These findings establish a role for SRA1 in the development of tomato trichomes and also implicate the actin-cytoskeleton network in cytosolic control of specialized metabolism for plant defense. We also show that the brittleness of hl mutant stems is associated with altered mechanical and cell morphological properties of stem tissue, and demonstrate that this defect is directly linked to the mutation in SRA1. PMID:27481446

  6. Tissue-Culture Method of Cloning Rubber Plants

    NASA Technical Reports Server (NTRS)

    Ball, E. A.

    1983-01-01

    Guayule plant, a high-yield rubber plant cloned by tissue-culture method to produce multiple new plants that mature quickly. By adjusting culture medium, excised shoot tip produces up to 50 identical guayule plants. Varying concentration of cytokinin, single excised tip produces either 1 or several (up to 50) new plants.

  7. Two leptin genes and a leptin receptor gene of female chub mackerel (Scomber japonicus): Molecular cloning, tissue distribution and expression in different obesity indices and pubertal stages.

    PubMed

    Ohga, Hirofumi; Matsumori, Kojiro; Kodama, Ryoko; Kitano, Hajime; Nagano, Naoki; Yamaguchi, Akihiko; Matsuyama, Michiya

    2015-10-01

    Leptin is a hormone produced by fat cells that regulates the amount of fat stored in the body and conveys nutritional status to the reproductive axis in mammals. In the present study we identified two subtypes of leptin genes (lepa and lepb) and a leptin receptor gene (lepr) from chub mackerel (Scomber japonicus) and there gene expression under different feeding conditions (control and high-feed) and pubertal development stages was analyzed using quantitative real-time PCR. The protein lengths of LepA, LepB and LepR were 161 amino acids (aa), 163 aa and 1149 aa, respectively and both leptin subtypes shared only 15% similarity in aa sequences. In pubertal females, lepa was expressed in the brain, pituitary gland, liver, adipose tissue and ovary; however, in adult (gonadal maturation after the second in the life) females, lepa was expressed only in the liver. lepb was expressed primarily in the brain of all fish tested and was expressed strongly in the adipose tissue of adults. lepr was characterized by expression in the pituitary. The high-feed group showed a high conditioning factor level; unexpectedly, hepatic lepa and brain lepr were significantly more weakly expressed compared with the control-feed group. Furthermore, the expression levels of lepa, lepb and lepr genes showed no significant differences between pre-pubertal and post-pubertal fish. On the other hand, pituitary fshβ and lhβ showed no significant differences between different feeding groups of pre-pubertal fish. In contrast, fshβ and lhβ expressed abundantly in the post-pubertal fish of control feed group. Based on these results, whether leptin plays an important role in the nutritional status and pubertal onset of chub mackerel remains unknown.

  8. Molecular cloning, characterization and tissue distribution of two ostrich β-defensins: AvBD2 and AvBD7.

    PubMed

    Lu, Shun; Peng, Kemei; Gao, Qishuang; Xiang, Min; Liu, Huazhen; Song, Hui; Yang, Keli; Huang, Haibo; Xiao, Ke

    2014-11-15

    Avian β-defensins (AvBDs) are a family of small antimicrobial peptides that play important roles in the innate immunity of birds. Herein, we report on two new ostrich AvBD genes, AvBD2 and AvBD7, which were isolated from the bone marrow of ostriches (Struthio camelus). The coding regions of ostrich AvBD2 and AvBD7 comprised 195 bp and 201bp, which encoded 64 and 66 amino acids, respectively. Homology analysis showed that ostrich AvBD2 had the highest similarity (up to 86%) with the swan goose (Anser cygnoides) AvBD2, while ostrich AvBD7 shared the highest similarity (up to 81%) with chicken AvBD7. Analysis of the codon-usage bias showed that the two ostrich AvBDs had different codon-usage patterns from other AvBDs. The two synthetic AvBD peptides exhibited antibacterial activities against both Gram-positive and Gram-negative bacteria, and these activities decreased significantly in the presence of 100mM NaCl (P<0.01). Real-time reverse transcription-polymerase chain reaction analysis showed that AvBD2 and AvBD7 were widely expressed at different levels in 17 different tissues. This is the first report of the nucleotide sequences of ostrich AvBDs. Further investigations of these two AvBDs may help us to gain new insights into the immune defense system of the ostrich and to make subsequent therapeutic use of ostrich defensins. PMID:25127671

  9. Molecular cloning of chicken aggrecan. Structural analyses.

    PubMed Central

    Chandrasekaran, L; Tanzer, M L

    1992-01-01

    The large, aggregating chondroitin sulphate proteoglycan of cartilage, aggrecan, has served as a generic model of proteoglycan structure. Molecular cloning of aggrecans has further defined their amino acid sequences and domain structures. In this study, we have obtained the complete coding sequence of chicken sternal cartilage aggrecan by a combination of cDNA and genomic DNA sequencing. The composite sequence is 6117 bp in length, encoding 1951 amino acids. Comparison of chicken aggrecan protein primary structure with rat, human and bovine aggrecans has disclosed both similarities and differences. The domains which are most highly conserved at 70-80% identity are the N-terminal domains G1 and G2 and the C-terminal domain G3. The chondroitin sulphate domain of chicken aggrecan is smaller than that of rat and human aggrecans and has very distinctive repeat sequences. It has two separate sections, one comprising 12 consecutive Ser-Gly-Glu repeats of 20 amino acids each, adjacent to the other which has 23 discontinuous Ser-Gly-Glu repeats of 10 amino acids each; this latter region, N-terminal to the former one, appears to be unique to chicken aggrecan. The two regions contain a total of 94 potential chondroitin sulphate attachment sites. Genomic comparison shows that, although chicken exons 11-14 are identical in size to the rat and human exons, chicken exon 10 is the smallest of the three species. This is also reflected in the size of its chondroitin sulphate coding region and in the total number of Ser-Gly pairs. The putative keratan sulphate domain shows 31-45% identity with the other species and lacks the repetitive sequences seen in the others. In summary, while the linear arrangement of specific domains of chicken aggrecan is identical to that in the aggrecans of other species, and while there is considerable identity of three separate domains, chicken aggrecan demonstrates unique features, notably in its chondroitin sulphate domain and its keratan sulphate

  10. Molecular cloning of a putative crustacean hyperglycemic hormone (CHH) isoform from extra-eyestalk tissue of the blue crab (Callinectes sapidus), and determination of temporal and spatial patterns of CHH gene expression.

    PubMed

    Zheng, Junying; Chen, Hsiang-Yin; Choi, Cheol Young; Roer, Robert D; Watson, R Douglas

    2010-11-01

    Crustacean hyperglycemic hormone (CHH) is a polypeptide neurohormone involved in regulation of multiple physiological processes. We report here the cloning from thoracic ganglia of the blue crab (Callinectes sapidus) a cDNA (CsCHH-2) encoding a putative CHH isoform (CsCHH-2). CsCHH-2 is structurally similar to a putative preproCHH (CsCHH-1) previously cloned from eyestalk ganglia of C. sapidus. The two preprohormones possess an identical signal peptide and CHH precursor related peptide, but differ in the mature CHH polypeptide. An analysis by RT-PCR of the tissue distribution of CsCHH-1 and CsCHH-2 revealed the former is restricted to eyestalk neural ganglia, while the latter is widely distributed among tissues. The type of CHH transcript present in eyestalk and thoracic ganglia did not vary as a function of the molt cycle. An assessment of transcript abundance in tissues of intermolt crabs showed the abundance of the CsCHH-1 transcript in eyestalk ganglia far exceeds the abundance of the CsCHH-2 transcript in extra-eyestalk tissue. An assessment of transcript abundance during a molt cycle showed CsCHH-1 transcript abundance in eyestalk ganglia was low during intermolt, rose during premolt, reaching a peak in D(3), then fell prior to molting, and remained low during postmolt. By contrast, CsCHH-2 transcript abundance in thoracic ganglia was low during intermolt, rose sharply during D(2), then dropped in D(3) and remained low during postmolt. The results are consistent with the hypothesis that CsCHH-1 and CsCHH-2 differ with respect to physiological function.

  11. Microorganisms in the gut of beetles: evidence from molecular cloning.

    PubMed

    Zhang, Ning; Suh, Sung-Oui; Blackwell, Meredith

    2003-11-01

    We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers.

  12. Molecular cloning and characterization of a cDNA encoding endonuclease from potato (Solanum tuberosum).

    PubMed

    Larsen, Knud

    2005-11-01

    A cDNA, StEN1, encoding a potato (Solanum tuberosum) endonuclease was cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 906 nucleotides encoding a protein of 302 amino acids, and with a calculated molecular mass of 34.4kDa and a Pi of 5.6. The deduced StEN1 protein contains a putative signal sequence of 25 amino acid residues. The StEN1 encoded protein shows substantial homology to both plant and fungal endonucleases isolated and cloned from other sources. The highest identity (73%) was observed with AgCEL I from celery, Apium graveolens, ZEN1 from Zinnia elegans (69%) and DSA6 from daylily, Hemerocallis (68%). RT-PCR expression analysis demonstrated that the potato StEN1 gene is constitutively expressed in potato, although minor differences in expression level in different tissues were observed. PMID:16323278

  13. Molecular cloning and sequencing of zeta-crystallin/quinone reductase cDNA from human liver.

    PubMed

    Gonzalez, P; Rao, P V; Zigler, J S

    1993-03-31

    Zeta-crystallin is an enzyme-crystallin highly expressed in the lens of some hystricomorph rodents and camels. It has been shown to have a novel NADPH: quinone oxidoreductase activity and is present at enzymatic levels in a variety of tissues from various mammals. We report here the cDNA cloning of zeta-crystallin from a human liver library. One clone with the complete open reading frame was obtained. Ten nucleotides of the 5' and 796 of the 3' nontranslated regions are present in the clone including two possible polyadenylation signals. The deduced amino acid sequence is 328 residues long with a calculated molecular mass of 34910 daltons and isoelectric point of 8.73. It shows 84% identity with the guinea pig protein.

  14. Molecular cloning and amino acid sequence of human 5-lipoxygenase

    SciTech Connect

    Matsumoto, T.; Funk, C.D.; Radmark, O.; Hoeoeg, J.O.; Joernvall, H.; Samuelsson, B.

    1988-01-01

    5-Lipoxygenase (EC 1.13.11.34), a Ca/sup 2 +/- and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B/sub 4/. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the /sup 32/P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite DNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A/sub 4/ hydrolase or Ca/sup 2 +/-binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approx. = 2700 nucleotides in leukocytes, lung, and placenta.

  15. Construction of chimeric vaccinia viruses by molecular cloning and packaging.

    PubMed Central

    Scheiflinger, F; Dorner, F; Falkner, F G

    1992-01-01

    Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology. Images PMID:1438247

  16. Molecular cloning and characterization of an inorganic pyrophosphatase from barley.

    PubMed

    Visser, K; Heimovaara-Dijkstra, S; Kijne, J W; Wang, M

    1998-05-01

    A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During inhibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.

  17. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  18. Molecular cloning and transcription expression of 3-dehydroecdysone 3α-reductase (3de 3α-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L.

    PubMed

    Yang, Hua-jun; Xin, Hu-hu; Lu, Yan; Cai, Zi-zheng; Wang, Mei-xian; Chen, Rui-Ting; Liang, Shuang; Singh, Chabungbam Orville; Kim, Jong-nam; Miao, Yun-gen

    2013-10-01

    Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, and reproduction etc. The decreased ecdysteroid in titre results from enhanced ecdysteroid inactivation reactions including the formation of 3-epiecdyson under ecdysone oxidase and 3-dehydroecdysone 3α-reductase (3DE 3α-reductase). In this paper, we cloned and characterized 3-dehydroecdysone 3α-reductase (3DE 3α-reductase) in different tissues and developing stage of the silkworm, Bombyx mori L. The B. mori 3DE 3α-reductase cDNA contains an ORF 783 bp and the deduced protein sequence containing 260 amino acid residues. Analysis showed the deduced 3DE 3α-reductase belongs to SDR family, which has the NAD(P)-binding domain. Using the Escherichia coli, a high level expression of a fusion polypeptide band of approx. 33 kDa was observed. High transcription of 3DE 3α-reductase was mainly presented in the midgut and hemolymph in the third day of fifth instar larvae in silkworm. The expression of 3DE 3α-reductase at different stages of larval showed that the activity in the early instar was high, and then reduced in late instar. This is parallel to the changes of molting hormone titer in larval. 3DE 3α-reductase is key enzyme in inactivation path of ecdysteroid. The data elucidate the regulation of 3DE 3α-reductase in ecdyteroid titer of its targeting organs and the relationship between the enzyme and metamorphosis. PMID:24038161

  19. Molecular cloning and characterisation of banana fruit polyphenol oxidase.

    PubMed

    Gooding, P S; Bird, C; Robinson, S P

    2001-09-01

    Polyphenol oxidase (PPO; EC 1.10.3.2) is the enzyme thought to be responsible for browning in banana [Musa cavendishii (AAA group, Cavendish subgroup) cv. Williams] fruit. Banana flesh was high in PPO activity throughout growth and ripening. Peel showed high levels of activity early in development but activity declined until ripening started and then remained constant. PPO activity in fruit was not substantially induced after wounding or treatment with 5-methyl jasmonate. Banana flowers and unexpanded leaf roll had high PPO activities with lower activities observed in mature leaves, roots and stem. Four different PPO cDNA clones were amplified from banana fruit (BPO1, BPO11, BPO34 and BPO35). Full-length cDNA and genomic clones were isolated for the most abundant sequence (BPO1) and the genomic clone was found to contain an 85-bp intron. Introns have not been previously found in PPO genes. Northern analysis revealed the presence of BPO1 mRNA in banana flesh early in development but little BPO1 mRNA was detected at the same stage in banana peel. BPO11 transcript was only detected in very young flesh and there was no detectable expression of BPO34 or BPO35 in developing fruit samples. PPO transcripts were also low throughout ripening in both flesh and peel. BPO1 transcripts were readily detected in flowers, stem, roots and leaf roll samples but were not detected in mature leaves. BPO11 showed a similar pattern of expression to BPO1 in these tissues but transcript levels were much lower. BPO34 and BPO35 mRNAs were only detected at a low level in flowers and roots and BPO34 transcript was detected in mature leaves, the only clone to do so. The results suggest that browning of banana fruit during ripening results from release of pre-existing PPO enzyme, which is synthesised very early in fruit development.

  20. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.

    PubMed

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective. PMID:23512843

  1. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics

    NASA Astrophysics Data System (ADS)

    Makhov, Dmitry V.; Glover, William J.; Martinez, Todd J.; Shalashilin, Dmitrii V.

    2014-08-01

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as "cloning," in analogy to the "spawning" procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, "trains," as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions.

  2. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics

    SciTech Connect

    Makhov, Dmitry V.; Shalashilin, Dmitrii V.; Glover, William J.; Martinez, Todd J.

    2014-08-07

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as “cloning,” in analogy to the “spawning” procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, “trains,” as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions.

  3. Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.

    PubMed Central

    Lawman, P; Bleiweis, A S

    1991-01-01

    We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 PMID:1938938

  4. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics.

    PubMed

    Makhov, Dmitry V; Glover, William J; Martinez, Todd J; Shalashilin, Dmitrii V

    2014-08-01

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as "cloning," in analogy to the "spawning" procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, "trains," as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions. PMID:25106573

  5. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    ERIC Educational Resources Information Center

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students a chance…

  6. Molecular model for hydrated biological tissues

    NASA Astrophysics Data System (ADS)

    Sato, Erika Tiemi; Rocha, Alexandre Reily; de Carvalho, Luis Felipe das Chagas e. Silva; Almeida, Janete Dias; Martinho, Herculano

    2015-06-01

    A density-functional microscopic model for soft tissues (STmod) is presented. The model was based on a prototype molecular structure from experimentally resolved type I collagen peptide residues and water clusters treated in periodic boundary conditions. We obtained the optimized geometry, binding and coupling energies, dipole moments, and vibrational frequencies. The results concerning the stability of the confined water clusters, the water-water, and water-collagen interactions were successfully correlated to some important experimental trends of normal and inflammatory tissues.

  7. Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

    PubMed

    Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

    1997-02-01

    Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

  8. Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata.

    PubMed

    Chase, M R; Raina, K; Bruno, J; Sugumaran, M

    2000-10-01

    Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (K(a)=40 microM) was achieved by the cationic detergent, cetyl pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserved C-terminal region common to all known PPOs.

  9. Cloning changes the response to obesity of innate immune factors in blood, liver, and adipose tissues in domestic pigs.

    PubMed

    Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan; Heegaard, Peter M H

    2013-06-01

    The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity.

  10. Molecular cloning and mRNA expression of peroxiredoxin gene in black tiger shrimp (Penaeus monodon).

    PubMed

    Qiu, Lihua; Ma, Zhuojun; Jiang, Shigui; Wang, Weifang; Zhou, Falin; Huang, Jianhua; Li, Jianzhu; Yang, Qibin

    2010-07-01

    The techniques of homology cloning and anchored PCR were used to clone the peroxiredoxin (Prx) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp Prx (PmPrx) contained a 5' untranslated region (UTR) of 51 bp, an ORF (open reading frame) of 582 bp encoding a polypeptide of 193 amino acids with an estimated molecular mass of 22.15 kDa and a 3' UTR of 948 bp. Sequence comparison showed that PmPrx shared higher identities with Prx IVs than that with other isoforms of Prx, indicating PmPrx was a member of the Prx IV family. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of PmPrx in different tissues and the temporal expression of PmPrx in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of PmPrx was detected in the tissues of hepatopancreas, gonad and heart. The expression of PmPrx in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that PmPrx was a constitutive and inducible expressed protein and could be induced by LPS.

  11. Molecular cloning and expression analysis of TRAF3 in chicken.

    PubMed

    Yang, H L; Feng, Z Q; Zeng, S Q; Li, S M; Zhu, Q; Liu, Y P

    2015-01-01

    Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a crucial regulator that suppresses c-Jun N-terminal kinase and non-canonical nuclear factor-kB signaling, but facilitates type I interferon production. To determine TRAF3 function in innate immune responses among birds, particularly chicken, we cloned and characterized the chicken TRAF3 gene (chTRAF3) and detected its tissue expression profile in chicken. We also detected the differential expression of chTRAF3 and its downstream gene interferon-β (IFN-β) upon different stimuli in primary chicken embryo fibroblast cells. Two chTRAF3 gene products, chTRAF3-1 and chTRAF3-2, can be produced by alternative splicing. The full-length coding sequence of chTRAF3 (chTRAF3-1) was 1704 base pairs and encoded a protein of 567 amino acids with high identity to TRAF3 homologs from mammals and other birds. The deduced amino acid sequence showed typical characteristics of TRAFs, with a RING finger domain, 2 zf-TRAF motifs, and a MATH domain. Quantitative real-time polymerase chain reaction analysis revealed broad expression of chTRAF3 in all detected tissues, with abundant expression in the spleen, thymus, lung, and small intestine. Expression of chTRAF3 was significantly upregulated in a time- and concentration-dependent manner in chicken embryo fibroblast cells challenged with poly I:C or poly dA-dT. Furthermore, chTRAF3 and IFN-β mRNA expression from chicken embryo fibroblast cells challenged with Newcastle disease virus F48E9 suffered intense suppression compared with Newcastle disease virus Mukteswar infection. Our results indicate that chTRAF3 plays important roles in defending against both RNA and DNA virus infection. PMID:25966214

  12. Molecular cloning and expression analysis of pig CD138.

    PubMed

    Bae, Joonbeom; Jeong, Seonah; Lee, Ju Yeon; Lee, Hyun-Jeong; Choi, Bong-Hwan; Kim, Ji-Eun; Choi, Inho; Chun, Taehoon

    2013-12-01

    CD138 (syndecan-1) interacts with various components of the extracellular matrix and associates with the actin cytoskeleton. In this study, we cloned pig CD138 cDNA and determined its complete cDNA sequence. Pig CD138 cDNA contained an open reading frame (930 bp) encoding 309 amino acids with five well conserved putative glycosaminoglycan attachment sites, a putative cleavage site for matrix metalloproteinases, and conserved motifs involved in signal transduction among mammalian species. Pig CD138 mRNA was detected in various tissues, including lymphoid and non-lymphoid organs, indicating the multicellular functions of CD138 in pigs. Western blot and flow cytometry analyses detected an approximate 35 kDa pig CD138 protein expressed on the cell surface. Further immunohistochemistry analysis revealed that CD138 expression was mainly observed in submucosa and lamina propria of the pig small intestine. Further study will be necessary to define the functional importance of CD138 during specific infectious diseases in pigs. PMID:24128845

  13. The 5-HT4 receptor: molecular cloning and pharmacological characterization of two splice variants.

    PubMed Central

    Gerald, C; Adham, N; Kao, H T; Olsen, M A; Laz, T M; Schechter, L E; Bard, J A; Vaysse, P J; Hartig, P R; Branchek, T A

    1995-01-01

    Molecular cloning efforts have provided primary amino acid sequence and signal transduction data for a large collection of serotonin receptor subtypes. These include five 5-HT1-like receptors, three 5-HT2 receptors, one 5-HT3 receptor, two 5-HT5 receptors, one 5-HT6 receptor and one 5-HT7 receptor. Molecular biological information on the 5-HT4 receptor is notably absent from this list. We now report the cloning of the pharmacologically defined 5-HT4 receptor. Using degenerate oligonucleotide primers, we identified a rat brain PCR fragment which encoded a '5-HT receptor-like' amino acid sequence. The corresponding full length cDNA was isolated from a rat brain cDNA library. Transiently expressed in COS-7 cells, this receptor stimulates adenylyl cyclase activity and is sensitive to the benzamide derivative cisapride. The response is also blocked by ICS-205930. Interestingly, we isolated two splice variants of the receptor, 5-HT4L and 5-HT4S, differing in the length and sequence of their C-termini. In rat brain, the 5-HT4S transcripts are restricted to the striatum, but the 5-HT4L transcripts are expressed throughout the brain, except in the cerebellum where it was barely detectable. In peripheral tissues, differential expression was also observed in the atrium of the heart where only the 5-HT4S isoform was detectable. Images PMID:7796807

  14. Molecular cloning and characterization of a unique type of human papillomavirus from an immune deficient patient.

    PubMed

    Ostrow, R S; Zachow, K R; Thompson, O; Faras, A J

    1984-04-01

    Several papillomas from a single patient who exhibited an unusual immune deficiency syndrome were analyzed for the presence of specific human papillomavirus (HPV) types. Preliminary analysis indicated that the HPV DNA species present in each of these tissues was quite unlike any of the previously characterized HPV types. In order to more rigorously analyze the HPV from this patient we have isolated the HPV DNA by molecularly cloning it into a bacteriophage lambda vector and have constructed a detailed restriction endonuclease map. Comparative hybridization studies using S1 nuclease analyses showed 6% or less nucleotide sequence homology of this viral DNA with HPV types 1, 2, 3, 4, 5, 6, or an HPV-11, molecularly cloned in this laboratory. Moreover, Southern blot analyses under stringent hybridization conditions revealed little, if any, hybridization to HPV types 1, 2, 4, 5, 7, 8, 10, 11, HPV-EV isolated from a patient with epidermodysplasia verruciformis (EV), or 2 previously described HPVs (HPV-P and HPV-PW) related to HPV-3. There was, however, a very weak sequence homology detected with HPV-6 and an extremely weak homology to HPV-3. No filter hybridization was observed with the recently characterized HPVs 9 or -12 to -24. These data accumulatively indicate that the HPV species from this immunosuppressed patient represents a new, hitherto unidentified HPV type. PMID:6323588

  15. Cloning higher plants from aseptically cultured tissues and cells

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  16. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay

    PubMed Central

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

    2016-01-01

    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF-7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot-based molecular targeted imaging techniques (which stained pan-cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF-7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot-based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology. PMID:27572664

  17. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

    PubMed

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

    2016-10-01

    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

  18. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

    PubMed

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

    2016-10-01

    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology. PMID:27572664

  19. Molecular model for hydrated biological tissues.

    PubMed

    Sato, Erika Tiemi; Rocha, Alexandre Reily; de Carvalho, Luis Felipe das Chagas e Silva; Almeida, Janete Dias; Martinho, Herculano

    2015-06-01

    A density-functional microscopic model for soft tissues (STmod) is presented. The model was based on a prototype molecular structure from experimentally resolved type I collagen peptide residues and water clusters treated in periodic boundary conditions. We obtained the optimized geometry, binding and coupling energies, dipole moments, and vibrational frequencies. The results concerning the stability of the confined water clusters, the water-water, and water-collagen interactions were successfully correlated to some important experimental trends of normal and inflammatory tissues. PMID:26172825

  20. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression.

    PubMed

    Unger, Tamar; Jacobovitch, Yossi; Dantes, Ada; Bernheim, Reut; Peleg, Yoav

    2010-10-01

    Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system. PMID:20600952

  1. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule

    SciTech Connect

    Dunne, P. Owen, M.J.; Hanby, A.M.; Poulsom, R.

    1995-11-20

    FAT, a new member of the human cadherin super-family, has been isolated from the T-leukemia cell line J6. The predicted protein closely resembles the Drosophila tumor suppressor fat, which is essential for controlling cell proliferation during Drosophila development. The gene has the potential to encode a large transmembrane protein of nearly 4600 residues with 34 tandem cadherin repeats, five EGF-like repeats, and a laminin A-G domain. The cytoplasmic sequence contains two domains with distant homology to the cadherin catenin-binding region. Northern blotting analysis of J6 mRNA demonstrated full-length, approximately 15-kb, FAT message in addition to several 5{prime}-truncated transcripts. In addition to its presence in J6 cells, in situ hybridization revealed FAT mRNA expression in epithelia and in some mesenchymal compartments. Furthermore, higher levels of expression were observed in fetal, as opposed to adult, tissue, suggesting that its expression may be developmentally regulated in these tissues. FAT shows homologies with a number of proteins important in developmental decisions and cell:cell communication and is the first fat-like protein reported in vertebrates. The gene encoding FAT was located by in situ hybridization on chromosome 4q34-q35. We propose that this family of molecules is likely to be important in mammalian developmental processes and cell communication. 80 refs., 10 figs., 1 tab.

  2. Molecular cloning and structural characterization of the R locus of maize: Annual progress report for period September 29, 1986-September 28, 1987

    SciTech Connect

    Dellaporta, S.L.

    1987-07-01

    Last year we reported on the isolation of a molecular clone of the R locus of maize using an Ac transposon tagging strategy. During the past year we have confirmed the identity of this clone and have begun a molecular analysis of several R alleles. Our main focus continues to be on the analysis of R-r, an allele containing both seed (S) and plant (P) components. Genomic blot analysis and gene cloning experiments suggest that the R-r allele may be organized as a triplication. In addition to (P) and (S), there appears to be a third cryptic component we refer to as (Q). We are attempting to clone the complete R-r allele by chromosome walking techniques to determine the molecular organization of R-r. The second objective of our research on R is to understand the mechanism of tissue-specific regulation of anthocyanin. We are characterizing several R alleles that condition different pigmentation patterns in plant and seed tissues. In order to determine the allelic differences among tissue-specific components we have obtained genomic clones and are performing DNA sequence analysis to regions of several tissue-specific components that may be responsible for these allelic differences. 1 ref.

  3. Molecular cloning and analysis of lymphokines. Volume 13

    SciTech Connect

    Webb, D.R.; Goeddel, D.V.

    1987-01-01

    These proceedings collect papers on the subject of lymphokines. Topics include: DNA-cloning of mouse and human lymphokine genes, inteferons, interleukins, gene expression, tumor necrosis factors, and recombinant DNA.

  4. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    PubMed

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue. PMID:27032955

  5. Gene expression analysis of the CD4+ T-cell clones derived from gingival tissues of periodontitis patients.

    PubMed

    Ito, H; Honda, T; Domon, H; Oda, T; Okui, T; Amanuma, R; Nakajima, T; Yamazaki, K

    2005-12-01

    The function of T cells infiltrating periodontitis lesions is complex and has not been fully elucidated. Here, we established T-cell clones from the gingival tissues of periodontitis patients and examined their gene expression. A total of 57 and 101 T-cell clones were established by means of immobilized anti-CD3 antibody and IL-2 from gingival tissues and peripheral blood, respectively. The gingival T-cell clones were derived from three patients, and the peripheral blood T-cell clones from two of these patients and a further patient whose gingival T-cell clones were not established. Gingival tissues were also obtained from a further 19 periodontitis patients. The expression of cytokines and molecules related to both regulatory function and tissue destruction were examined by means of reverse-transcription polymerase chain reaction. All the gingival T-cell clones expressed mRNA for TGF-beta1, CTLA-4, and CD25, and all the T-cell clones from peripheral blood expressed IFN-gamma and TGF-beta1 mRNAs. Most but not all the T-cell clones from gingival tissues and peripheral blood expressed mRNA for IFN-gamma and, CD25 and CTLA-4, respectively. The frequency of T-cell clones and gingival tissues expressing FOXP3, a possible master gene for mouse CD4(+)CD25(+) regulatory T cells, was very high (97%, 93%, and 100% for gingival T-cell clones, peripheral blood T-cell clones, and gingival tissues, respectively). Whereas the frequency of IL-4-expressing T-cell clones was lower for gingival T-cell clones (70% vs. 87%), the frequency of the gingival T-cell clones expressing IL-10 and IL-17 was higher than peripheral blood T-cell clones (75% vs. 62% for IL-10, 51% vs. 11% for IL-17). A similar expression profile was observed for gingival T-cell clones compared with gingival tissue samples with the exception of IL-4 expression, where the frequency of positive samples was lower in the gingival tissues (70% vs. 11%). These results suggest that the individual T cells infiltrating

  6. Plant tissue culture and molecular markers.

    PubMed

    Tamayo-Ordoñez, María; Huijara-Vasconselos, Javier; Quiroz-Moreno, Adriana; Ortíz-García, Matilde; Sánchez-Teyer, Lorenzo Felipe

    2012-01-01

    Tissue culture can be used to propagate elite material or to generate new variability by employing somaclonal variation. Genetic stability of the process must be evaluated analyzing DNA profiles by the use of molecular markers. Several techniques have been reported for the screening of genetic variation on tissue culture derived material; however, a highly informative and good relation among the time-cost-information is obtained using Amplified Fragment Length Polymorphism (AFLP) in automatic sequencer. This technique involves a double-digestion of DNA with restriction enzymes, ligation of adapters at both extremities of the restriction fragments, and finally, selective polymerase chain reaction (PCR) amplification of the fragments. A semiautomatic process for the analysis could be used, but several considerations must be taken into account before such a use. PMID:22610640

  7. Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

    PubMed

    Knight, C; Simmons, D; Gu, T T; Gluhak-Heinrich, J; Pavlin, D; Zeichner-David, M; MacDougall, M

    2001-10-01

    Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis.

  8. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene

    PubMed Central

    Sadeghi, H. Mir Mohammad; Ahmadi, R.; Aghaabdollahian, S.; Mofid, M.R.; Ghaemi, Y.; Abedi, D.

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities. PMID:22110522

  9. Cloning, expression, and regulation of tissue-specific genes in Drosophila

    SciTech Connect

    Korochkin, L.I.

    1995-08-01

    The family of esterase genes was studied in various Drosophilia species. These genes are classified as tissue-specific and housekeeping ones. The expression of tissue-specific esterases in the male reproductive system of Drosophilia species from the virilis and melanogaster groups was thoroughly examined. Modifier genes controlling activity level, time of synthesis, and distribution in cells of the tissue-specific esterase isozyme from the ejaculatory bulb were revealed. The structural gene coding of this enzyme was isolated, cloned, and sequenced. This gene was shown to be similar in different Drosophilia species; the transcriptional level of tissue specificity of this gene was determined. The possibility of transformating the tissue-specific gene into a housekeeping one was demonstrated. In different Drosophilia species, this gene can be expressed in different parts of the reproductive system. In transgenic males carrying the gene of another species, the foreign gene is expressed as in the donor. 68 refs., 11 figs.

  10. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    ERIC Educational Resources Information Center

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  11. Spontaneous aneuploidy and clone formation in adipose tissue stem cells during different periods of culturing.

    PubMed

    Buyanovskaya, O A; Kuleshov, N P; Nikitina, V A; Voronina, E S; Katosova, L D; Bochkov, N P

    2009-07-01

    Cytogenetic analysis of 13 mesenchymal stem cell cultures isolated from normal human adipose tissue was carried out at different stages of culturing. The incidence of chromosomes 6, 8, 11, and X aneuploidy and polyploidy was studied by fluorescent in situ hybridization. During the early passages, monosomal cells were more often detected than trisomal ones. A clone with chromosome 6 monosomy was detected in three cultures during late passages.

  12. Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase

    SciTech Connect

    Holst, L.S.; Laurell, H.; Holm, C.

    1996-08-01

    By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSL{sub tes}. Due to an addition of amino acids at the NH{sub 2}-termini, rat and human HSL{sub tes} consist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSL{sub adi}). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSL{sub adi}. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSL{sub adi} sequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M{sub r} {approximately}120,000) that exhibited catalytic activity similar to that of HSL{sub adi}. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells. 34 refs., 5 figs.

  13. Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos.

    PubMed

    Park, Mi-Ryung; Im, Gi-Sun; Kim, Sung Woo; Hwang, Seongsoo; Park, Jae-Hong; Kim, Hyun; Do, Yoon Jung; Park, Soo Bon; Yang, Bo-Suck; Song, Young Min; Cho, Jae-Hyeon; Ko, Yeoung-Gyu

    2013-06-01

    The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.97±2.3 cloned fetuses were successfully recovered from three surrogates at gestational day 30. An analysis of the 6.97±2.3 cloned embryos revealed that most had severe extraembryonic defects. The extraembryonic tissue from the SCNT embryos was abnormally small compared with that of the control. To investigate the differentially expressed genes between the SCNT and control extraembryonic tissues, we compared the gene expression profiles of the extraembryonic tissues from gestational day 30 cloned pig embryos with those from the control using an annealing control primer-based GeneFishing polymerase chain reaction. As a result, we found that a total of 50 genes were differentially expressed by utilizing 120 ACPs, 38 genes of which were known. Among them, 26 genes were up-regulated, whereas 12 genes were down-regulated. Real-time RT-PCR showed that apoptosis-related genes were expressed significantly higher in SCNT extraembryonic tissue than in the control, whereas metabolism-related genes were expressed at significantly lower levels in the SCNT extraembryonic tissue. These observations strongly indicate that early gestational death of SCNT embryo is caused, at least in part, by the disruption of developing extraembryonic tissues as a result of aberrant gene expression, which results in abnormal apoptosis and metabolism.

  14. Molecular cloning and functional characterization of a putative sulfite oxidase (SO) ortholog from Nicotiana benthamiana.

    PubMed

    Xia, Zongliang; Su, Xinhong; Wu, Jianyu; Wu, Ke; Zhang, Hua

    2012-03-01

    Sulfite oxidase (SO) catalyzes the oxidation of sulfite to sulfate and thus has important roles in diverse metabolic processes. However, systematic molecular and functional investigations on the putative SO from tobacco (Nicotiana benthamiana) have hitherto not been reported. In this work, a full-length cDNA encoding putative sulfite oxidase from N. benthamiana (NbSO) was isolated. The deduced NbSO protein shares high homology and typical structural features with other species SOs. Phylogenetic analysis indicates that NbSO cDNA clone encodes a tobacco SO isoform. Southern blot analysis suggests that NbSO is a single-copy gene in the N. benthamiana genome. The NbSO transcript levels were higher in aerial tissues and were up-regulated in N. benthamiana during sulfite stress. Reducing the SO expression levels through virus-induced gene silencing caused a substantial accumulation in sulfite content and less sulfate accumulation in N. benthamiana leaves when exposed to sulfite stress, and thus resulted in decreased tolerance to sulfite stress. Taken together, this study improves our understanding on the molecular and functional properties of plant SO and provides genetic evidence on the involvement of SO in sulfite detoxification in a sulfite-oxidizing manner in N. benthamiana plants. PMID:21667106

  15. Molecular cloning of seal myoglobin mRNA.

    PubMed Central

    Wood, D; Blanchetot, A; Jeffreys, A J

    1982-01-01

    Grey seal skeletal muscle containing high levels of myoglobin was used to prepare poly(A)+ RNA. In vitro translation of this RNA produced a range of polypeptides including myoglobin. cDNA was prepared by reverse transcription of muscle poly(A)+ RNA and cloned into the plasmid pAT 153. 4% of cDNA recombinants were shown to contain myoglobin cDNA inserts. DNA sequence analysis of one clone (pSM 178) which contained a relatively large myoglobin cDNA insert showed an incomplete cDNA comprising the terminal 293 nucleotides of 3' non-translated mRNA sequences. Hybridization experiments using this myoglobin cDNA indicated that seal myoglobin is coded by a single gene which is transcribed to give a 1400 nucleotide mRNA considerably longer than related haemoglobin mRNAs. Images PMID:6185919

  16. Tissue engineering, stem cells, cloning, and parthenogenesis: new paradigms for therapy

    PubMed Central

    Hipp, Jason; Atala, Anthony

    2004-01-01

    Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs which is worsening yearly due to the aging population. Scientists in the field of tissue engineering apply the principles of cell transplantation, materials science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. The present article reviews recent progress in tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure. PMID:15588286

  17. Molecular cloning of the gene for human anti-haemophilic factor IX.

    PubMed

    Choo, K H; Gould, K G; Rees, D J; Brownlee, G G

    1982-09-01

    A functional deficiency of factor IX, one of the coagulation factors involved in blood clotting, leads to the bleeding disorder known as Christmas disease, or haemophilia B. Both this disease and haemophilia A (factor VIII (C) deficiency) are X chromosome-linked and together occur at a frequency of approximately 1 in 10,000 males. The molecular basis for the functional alteration of factor IX in Christmas disease is not clearly understood. As a first step towards the elucidation of the molecular events involved, we have attempted molecular cloning of the factor IX gene. We used a bovine factor IX cDNA clone, isolated using synthetic oligonucleotides as probes, to screen a cloned human gene library. Here we report the isolation and partial characterization of a lambda recombinant phage containing the human factor IX gene.

  18. Human phenol sulfotransferase STP2 gene: Molecular cloning, structural characterization, and chromosomal localization

    SciTech Connect

    Her, C.; Raftogianis, R.; Weinshilboum, R.M.

    1996-05-01

    Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of {open_quotes}simple{close_quotes} planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs, STP2, as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans. STP2 spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of most STP2 exon-intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans, STM; a rat PST gene; a human estrogen ST (EST) gene, STE; and a guinea pig EST gene. The two initial STP2 exons, IA and IB, were identified by performing 5{prime}-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5{prime}untranslated region sequences. The two apparent 5{prime}-ons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements. STP2 was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization of STP2 will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissues. 63 refs., 7 figs., 1 tab.

  19. Semiautomated clone verification by real-time PCR using molecular beacons.

    PubMed

    van Schie, R C; Marras, S A; Conroy, J M; Nowak, N J; Catanese, J J; de Jong, P J

    2000-12-01

    Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.

  20. Molecular cloning and expression of the human interleukin 5 receptor

    PubMed Central

    1992-01-01

    Human interleukin 5 (IL-5) plays an important role in proliferation and differentiation of human eosinophils. We report the isolation of cDNA clones from cDNA libraries of human eosinophils by using murine IL-5 receptor alpha chain cDNA as a probe. Analysis of the predicted amino acid sequence indicated that the human IL-5 receptor has approximately 70% amino acid sequence homology with the murine IL-5 receptor and retains features common to the cytokine receptor superfamily. One cDNA clone encodes a glycoprotein of 420 amino acids (Mr 47,670) with an NH2- terminal hydrophobic region (20 amino acids), a glycosylated extracellular domain (324 amino acids), a transmembrane domain (21 amino acids), and a cytoplasmic domain (55 amino acids). Another cDNA encodes only the extracellular domain of this receptor molecule. Other cDNA clones encode molecules having diversified cytoplasmic domains. COS7 cells transfected with the cDNA expressed a approximately 60-kD protein and bound IL-5 with a single class of affinity (Kd = 250-590 pM). The Kd values were similar to that observed in normal human eosinophils. In contrast to the murine 60-kD alpha chain, which binds IL-5 with low affinity (Kd = approximately 10 nM), the human alpha chain homologue can bind IL-5 with much higher affinity by itself. RNA blot analysis of human cells demonstrated two transcripts (approximately 5.3 and 1.4 kb). Both of them were expressed in normal human eosinophils and in erythroleukemic cell line TF-1, which responds to IL-5. The human IL-5 receptor characterized in this paper is essential for signal transduction, because expression of this molecule in murine IL-3-dependent cell line FDC-P1 allowed these cells to proliferate in response to IL-5. PMID:1732409

  1. Molecular Cloning of MAPK Gene Family Using Synthetic Oligonucleotide Probe.

    PubMed

    Zhou, Song; Wang, Qin; Chen, Jing; Chen, Jiang-Ye

    1999-01-01

    MAPK(mitogen activated protein kinase) is a kind of Ser/Thr protein kinase. The MAPKs play an important role in several different signal transduction pathways. The MAPKs may also have a role in morphorgenesis of Candida albicans. An oligonucleotide probe was used to screen novel MAPKs in C. albicans. All MAPKs shared high homogeneity in their eleven kinase subdomains, especially subdoman VII and VIII. In subdomain VII, nearly all MAPKs have the same KIDFGLAR sequence, and the two known MAPKs in C. albicans CEK1 and MKC1 have only one different nucleotide in that DNA sequence. This probe was hybridized with C. albicans genomic DNA. Under stringent conditions, the probe could only hybridize with CEK1 and MKC1 gene fragment. But when hybridized at 40 degrees in non-SDS solution, two novel bands appeared. This condition was used to screen SC5314 DNA library, and many positive clones with different hybridization density were obtained. The strongest hybridization clones were identified to contain CEK1 and MKC1 gene. From the stronger positive hybridization clones, two novel genes were identified. The first gene, named CRK1(CDC2-related protein kinase 1), shared high homogeneity to MAPKs, but was not of them. It is closest to SGV1 from S. cerevisiae (with homology 47%) and PITALRE from human (with homology 41%), both of which are CDC2-related protein kinases. The second gene called CEK2(Candida albicans extracelluar signal-regulated kinase 2) is a novel MAPK of Candida albicans, which shares the highest identity with CEK1 and its S. cerevisiae homologs, FUS3 and KSS1, two redundant MAPKs in yeast pheromone response and morphogenesis. PMID:12114967

  2. Molecular cloning of human ornithine aminotransferase mRNA

    SciTech Connect

    Inana, G.; Totsuka, S.; Redmond, M.; Dougherty, T.; Nagle, J.; Shiono, T.; Ohura, T. Kominami, E.; Katunuma, N.

    1986-03-01

    The isolation and characterization of a cDNA clone for the mRNA of human ornithine aminotransferase (OATase; ornithine-oxo-acid aminotransferase; L-ornithine:2-oxo-acid aminotransferase, EC 2.6.1.13), a nonabundant mitochondrial matrix enzyme that is severely deficient in a hereditary chorioretinal degenerative disease (gyrate atrophy), is described. Human liver, retina, and retinoblastoma (Y79) mRNAs were prepared and tested for the OATase mRNA content by in vitro translation, immunoprecipitation, and NaDodSO/sub 4//PAGE. The retinoblastoma cells were found to be expressing this enzyme at a relatively high level. The primary translation product of the OATase mRNA is larger than the pure OATase protein on NaDodSO/sub 4//PAGE. lambdagt11 cDNA libraries were prepared from the human mRNAs, and the recombinant clones were immunoscreened as plaques with two different preparations of rabbit anti-human OATase antibodies. The amino acid sequences of seven tryptic peptides (115 amino acid residues) of the pure human OATase were obtained by microsequencing. When the tryptic peptide and cDNA-derived amino acid sequences were compared, homologies in 111 of 115 residues, including a match of 20 consecutive residues, were observed. An RNA blot hybridization of /sup 32/P-labeled OATase cDNA to normal human retina and retinoblastoma mRNAs demonstrated an OATase mRNA species of approx. = 2.2 kilobases.

  3. Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning.

    PubMed

    Arias, A; Lázaro, E; Escarmís, C; Domingo, E

    2001-05-01

    The mutant spectrum of a virus quasispecies in the process of fitness gain of a debilitated foot-and-mouth disease virus (FMDV) clone has been analysed. The mutant spectrum was characterized by nucleotide sequencing of three virus genomic regions (internal ribosome entry site; region between the two AUG initiation codons; VP1-coding region) from 70 biological clones (virus from individual plaques formed on BHK-21 cell monolayers) and 70 molecular clones (RT--PCR products cloned in E. coli). The biological and molecular clones provided statistically indistinguishable definitions of the mutant spectrum with regard to the distribution of mutations among the three genomic regions analysed and with regard to the types of mutations, mutational hot-spots and mutation frequencies. Therefore, the molecular cloning procedure employed provides a simple protocol for the characterization of mutant spectra of viruses that do not grow in cell culture. The number of mutations found repeated among the clones analysed was higher than expected from the mean mutation frequencies. Some components of the mutant spectrum reflected genomes that were dominant in the prior evolutionary history of the virus (previous passages), confirming the presence of memory genomes in virus quasispecies. Other components of the mutant spectrum were genomes that became dominant at a later stage of evolution, suggesting a predictive value of mutant spectrum analysis with regard to the outcome of virus evolution. The results underline the observation that greater insight into evolutionary processes of viruses may be gained from detailed clonal analyses of the mutant swarms at the sequence level.

  4. Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

    PubMed Central

    Hanner, M; Moebius, F F; Flandorfer, A; Knaus, H G; Striessnig, J; Kempner, E; Glossmann, H

    1996-01-01

    Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755605

  5. Cloning crops in a CELSS via tissue culture: Prospects and problems

    NASA Technical Reports Server (NTRS)

    Carman, John G.; Hess, J. Richard

    1990-01-01

    Micropropagation is currently used to clone fruits, nuts, and vegetables and involves controlling the outgrowth in vitro of basal, axillary, or adventitious buds. Following clonal multiplication, shoots are divided and rooted. This process has greatly reduced space and energy requirements in greenhouses and field nurseries and has increased multiplication rates by greater than 20 fold for some vegetatively propagated crops and breeding lines. Cereal and legume crops can also be cloned by tissue culture through somatic embryogenesis. Somatic embryos can be used to produce 'synthetic seed', which can tolerate desiccation and germinate upon rehydration. Synthetic seed of hybrid wheat, rice, soybean and other crops could be produced in a controlled ecological life support system. Thus, yield advantages of hybreds over inbreds (10 to 20 percent) could be exploited without having to provide additional facilities and energy for parental-line and hybrid seed nurseries.

  6. T-cell receptor heterogeneity of gamma delta T-cell clones from human female reproductive tissues.

    PubMed

    Christmas, S E; Brew, R; Deniz, G; Taylor, J J

    1993-03-01

    gamma delta T cells were isolated from human decidua parietalis, decidua basalis and cervix and cloned in the presence of interleukin-2 (IL-2). T-cell receptor (TcR) expression was then analysed and compared with that of a panel of gamma delta T-cell clones from peripheral blood. Only 17/40 (42.5%) clones from decidua parietalis were V gamma 9+/V delta 2+ as compared to 68/94 (72%) of peripheral blood clones (P < 0.005). Conversely, 50% of clones from decidua parietalis but only 15% of clones from peripheral blood were V delta 1+ (P < 0.001). At least seven distinct TcR types were identified among the panel of clones from decidua parietalis and at least six different types were expressed by the panel of 17 clones from cervix. This receptor heterogeneity was not a result of interdonor variation as in all instances where more than one clone was obtained from a single sample, individual clones having between two and five receptor types were identified. However, 23/24 (95.8%) of clones from decidua basalis were V gamma 9+/V delta 2+. Most clones from decidua parietalis and cervix, whether V gamma 9+/V delta 2+ or V delta 1+, were positive for the mucosal lymphocyte marker, HML-1, but expression was often heterogeneous within a single clone. In contrast, almost all gamma delta T-cell clones from peripheral blood were HML-1-. Thus, unlike the mouse, gamma delta T cells within these human female reproductive tissues have a diverse TcR repertoire which, in decidua parietalis, is distinct from that of peripheral blood.

  7. Molecular cloning and characterization of chitinase genes from Candida albicans.

    PubMed Central

    McCreath, K J; Specht, C A; Robbins, P W

    1995-01-01

    Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition. Images Fig. 2 Fig. 5 PMID:7708682

  8. Molecular transport in collagenous tissues measured by gel electrophoresis.

    PubMed

    Hunckler, Michael D; Tilley, Jennifer M R; Roeder, Ryan K

    2015-11-26

    Molecular transport in tissues is important for drug delivery, nutrient supply, waste removal, cell signaling, and detecting tissue degeneration. Therefore, the objective of this study was to investigate gel electrophoresis as a simple method to measure molecular transport in collagenous tissues. The electrophoretic mobility of charged molecules in tissue samples was measured from relative differences in the velocity of a cationic dye passing through an agarose gel in the absence and presence of a tissue section embedded within the gel. Differences in electrophoretic mobility were measured for the transport of a molecule through different tissues and tissue anisotropy, or the transport of different sized molecules through the same tissue. Tissue samples included tendon and fibrocartilage from the proximal (tensile) and distal (compressive) regions of the bovine flexor tendon, respectively, and bovine articular cartilage. The measured electrophoretic mobility was greatest in the compressive region of the tendon (fibrocartilage), followed by the tensile region of tendon, and lowest in articular cartilage, reflecting differences in the composition and organization of the tissues. The anisotropy of tendon was measured by greater electrophoretic mobility parallel compared with perpendicular to the predominate collagen fiber orientation. Electrophoretic mobility also decreased with increased molecular size, as expected. Therefore, the results of this study suggest that gel electrophoresis may be a useful method to measure differences in molecular transport within various tissues, including the effects of tissue type, tissue anisotropy, and molecular size.

  9. Molecular cloning, expression and characterization of acylpeptide hydrolase in the silkworm, Bombyx mori.

    PubMed

    Fu, Ping; Sun, Wei; Zhang, Ze

    2016-04-10

    Acylpeptide hydrolase (APH) can catalyze the release of the N-terminal amino acid from acetylated peptides. There were many documented examples of this enzyme in various prokaryotic and eukaryotic organisms. However, knowledge about APH in insects still remains unknown. In this study, we cloned and sequenced a putative silkworm Bombyx mori APH (BmAPH) gene. The BmAPH gene encodes a protein of 710 amino acids with a predicted molecular mass of 78.5kDa. The putative BmAPH and mammal APHs share about 36% amino acid sequence identity, yet key catalytic residues are conserved (Ser566, Asp654, and His686). Expression and purification of the recombinant BmAPH in Escherichia coli showed that it has acylpeptide hydrolase activity toward the traditional substrate, Ac-Ala-pNA. Furthermore, organophosphorus (OP) insecticides, chlorpyrifos, phoxim, and malathion, significantly inhibited the activity of the APH both in vitro and in vivo. In addition, BmAPH was expressed in all tested tissues and developmental stages of the silkworm. Finally, immunohistochemistry analysis showed that BmAPH protein was localized in the basement membranes. These results suggested that BmAPH may be involved in enhancing silkworm tolerance to the OP insecticides. In a word, our results provide evidence for understanding of the biological function of APH in insects.

  10. Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

    PubMed

    Yang, Yumei; Luo, Zhu; Zhang, Mengru; Liu, Chang; Gong, Ming; Zou, Zhurong

    2016-04-01

    H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi. PMID:26643082

  11. Molecular cloning and structural characterization of Ecdysis Triggering Hormone from Choristoneura fumiferana.

    PubMed

    P, Bhagath Kumar; K, Kasi Viswanath; S, Tuleshwori Devi; R, Sampath Kumar; Doucet, Daniel; Retnakaran, Arthur; Krell, Peter J; Feng, Qili; Ampasala, Dinakara Rao

    2016-07-01

    At the end of each stadium, insects undergo a precisely orchestrated process known as ecdysis which results in the replacement of the old cuticle with a new one. This physiological event is necessary to accommodate growth in arthropods since they have a rigid chitinous exoskeleton. Ecdysis is initiated by the direct action of Ecdysis Triggering Hormones on the central nervous system. Choristoneura fumiferana is a major defoliator of coniferous forests in Eastern North America. It is assumed that, studies on the ecdysis behavior of this pest might lead to the development of novel pest management strategies. Hence in this study, the cDNA of CfETH was cloned. The open reading frame of the cDNA sequence was found to encode three putative peptides viz., Pre-Ecdysis Triggering Hormone (PETH), Ecdysis Triggering Hormone (ETH), and Ecdysis Triggering Hormone Associated Peptide (ETH-AP). The CfETH transcript was detected in the epidermal tissue of larval and pupal stages, but not in eggs and adults. In order to explore the structural conformation of ETH, ab initio modelling and Molecular Dynamics (MD) Simulations were performed. Further, a library of insecticides was generated and virtual screening was performed to identify the compounds displaying high binding capacity to ETH. PMID:27012894

  12. Molecular cloning and daily variations of the Period gene in a reef fish Siganus guttatus.

    PubMed

    Park, Ji-Gweon; Park, Yong-Ju; Sugama, Nozomi; Kim, Se-Jae; Takemura, Akihiro

    2007-04-01

    As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus--a reef fish with a definite lunar-related rhythmicity--we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00 h and decreased at 18:00 h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.

  13. Molecular cloning and characterization of a galectin-1 homolog in orange-spotted grouper, Epinephelus coioides.

    PubMed

    Chen, Xiuli; Wei, Jingguang; Xu, Meng; Yang, Min; Li, Pingfei; Wei, Shina; Huang, Youhua; Qin, Qiwei

    2016-07-01

    As a member of animal lectin family, galectin has the functions of pathogen recognition, anti-bacteria and anti-virus. In the present study, a galectin-1 homolog (EcGel-1) from grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcGel-1 is 504 bp, including a 408 bp open reading frame (ORF) which encodes 135 amino acids with a molecular mass of 15.19 kDa. Quantitative real-time PCR analysis indicated that EcGel-1 was constitutively expressed in all analyzed tissues of healthy grouper. The expression of EcGel-1 in the spleen of grouper was differentially up-regulated challenged with Singapore grouper iridovirus (SGIV), poly (I:C), and LPS. EcGel-1 was abundantly distributed in the cytoplasm in GS cells. Recombinant EcGel-1(rEcGel-1) protein can make chicken erythrocyte aggregation, and combine with gram negative bacteria and gram positive bacteria in the presence of 2-Mercaptoethanol (β-ME). Taken together, the results showed that EcGel-1 may be an important molecule involved in pathogen recognition and pathogen elimination in the innate immunity of grouper.

  14. Molecular cloning and characterization of a Bombyx mori gene encoding the transcription factor Atonal.

    PubMed

    Hu, Ping; Feng, Fan; Xia, Hengchuan; Chen, Liang; Yao, Qin; Chen, Keping

    2014-01-01

    The atonal genes are an evolutionarily conserved group of genes encoding regulatory basic helix-loop-helix (bHLH) transcription factors. These transcription factors have a critical antioncogenic function in the retina, and are necessary for cell fate determination through the regulation of the cell signal pathway. In this study, the atonal gene was cloned from Bombyx mori, and the transcription factor was named BmAtonal. Sequence analysis showed that the BmAtonal protein shares extensive homology with other invertebrate Atonal proteins with the bHLH motif. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses revealed that BmAtonal was expressed in all developmental stages of B. mori and various larval tissues. The BmAtonal protein was expressed in Escherichia coli, and polyclonal antibodies were raised against the purified protein. By immunofluorescence, the BmAtonal protein was localized to both the nucleus and cytoplasm of BmN cells. After knocking out nuclear localization signals (NLS), the BmAtonal protein was only detected in the cytoplasm. In addition, using the B. mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system, the recombinant BmAtonal protein was successfully expressed in the B. mori cell line BmN. This work lays the foundation for exploring the biological functions of the BmAtonal protein, such as identifying its potential binding partners and understanding the molecular control of the formation of sensory organs. PMID:24873037

  15. Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

    PubMed

    Yang, Yumei; Luo, Zhu; Zhang, Mengru; Liu, Chang; Gong, Ming; Zou, Zhurong

    2016-04-01

    H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi.

  16. Mapping, cDNA cloning and tissue expression of the porcine thyrotropin-releasing hormone receptor gene.

    PubMed

    Jiang, Xiaoling; Cai, Zhaowei; Zhao, Xiaofeng; Zhang, Lifan; Chen, Zhe; Wang, Ying; Guo, Xiaoling; Xu, Ningying

    2011-01-01

    Thyrotropin-releasing hormone receptor (TRHR) is a G-protein-coupled receptor that plays a crucial role in regulating the hypothalamic-pituitary-thyroid axis by conveying the action of the hypothalamic tripeptide TRH, which is the primary central activator of this hormonal cascade. In the present study, the porcine TRHR (pTRHR) gene was localized to chromosome 4 by Radiation hybrid mapping. Quantitative trait loci affecting average backfat thickness, daily gain, and carcass and meat quality traits have been mapped to the region containing this gene. Further, the full-length cDNA of pTRHR was cloned and sequenced. pTRHR contains an open reading frame encoding 398 amino acids and shares 96.2% amino acid identity to human TRHR. Real-time quantitative RT-PCR showed that the mRNA of pTRHR is expressed in a variety of tissues, with high expression in the brain, hypothalamus, pituitary, testis, and fat tissue. The considerable expression level of TRHR mRNA found in fat tissue indicates potential direct action of TRH on lipocyte might exist. Additionally, two alternative spliced transcript variants of pTRHR were also isolated in this study. Our data provided basic molecular information which will be useful for further investigation on pTRHR gene.

  17. [Molecular cloning and structural characteristics of the R complex of maize]. Annual progress report

    SciTech Connect

    Not Available

    1992-07-01

    Studies on the R complex in Maize continued Progress is discussed in the following areas: Establishing identity of R components and cloning of R components; CO allele origin; molecular organization of R-r complex; NCO allele origin; genetic analysis of R-r complex; studies of the Sn locus and reverse paramutation.

  18. (Molecular cloning and structural characteristics of the R complex of maize)

    SciTech Connect

    Not Available

    1992-01-01

    Studies on the R complex in Maize continued Progress is discussed in the following areas: Establishing identity of R components and cloning of R components; CO allele origin; molecular organization of R-r complex; NCO allele origin; genetic analysis of R-r complex; studies of the Sn locus and reverse paramutation.

  19. Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase).

    PubMed

    Bonthron, D T; Brady, N; Donaldson, I A; Steinmann, B

    1994-09-01

    Essential fructosuria is one of the oldest known inborn errors of metabolism. It is a benign condition which is believed to result from deficiency of hepatic fructokinase (ketohexokinase, KHK, E.C.2.7.1.3). This enzyme catalyses the first step of metabolism of dietary fructose, conversion of fructose to fructose-1-phosphate. Despite the early recognition of this disorder, the primary structure of human KHK and the molecular basis of essential fructosuria have not been previously defined. In this report, the isolation and sequencing of full-length cDNA clones encoding human ketohexokinase are described. Alternative mRNA species and alternative KHK isozymes are produced by alternative polyadenylation and splicing of the KHK gene. The KHK proteins show a high level of sequence conservation relative to rat KHK. Direct evidence that mutation of the KHK structural gene is the cause of essential fructosuria was also obtained. In a well-characterized family, in which three of eight siblings have fructosuria, all affected individuals are compound heterozygotes for two mutations Gly40Arg and Ala43Thr. Both mutations result from G-->A transitions, and each alters the same conserved region of the KHK protein. Neither mutation was seen in a sample of 52 unrelated control individuals. An additional conservative amino acid change (Val49IIe) was present on the KHK allele bearing Ala43Thr.

  20. Molecular cloning of a hyaluronidase from Bothrops pauloensis venom gland

    PubMed Central

    2014-01-01

    Background Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victim’s body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silico analysis of the first hyaluronidase-like proteins from a Brazilian snake venom. Methods The cDNA sequence of hyaluronidase was cloned from the transcriptome of Bothrops pauloensis venom glands. This sequence was submitted to multiple alignment with other related sequences by ClustalW. A phylogenetic analysis was performed using MEGA 4 software by the neighbor joining (NJ) method. Results The cDNA from Bothrops pauloensis venom gland that corresponds to hyaluronidase comprises 1175 bp and codifies a protein containing 194 amino acid residues. The sequence, denominated BpHyase, was identified as hyaluronidase-like since it shows high sequence identities (above 83%) with other described snake venom hyaluronidase-like sequences. Hyaluronidases-like proteins are thought to be products of alternative splicing implicated in deletions of central amino acids, including the catalytic residues. Structure-based sequence alignment of BpHyase to human hyaluronidase hHyal-1 demonstrates a loss of some key secondary structures. The phylogenetic analysis indicates an independent evolution of BpHyal when compared to other hyaluronidases. However, these toxins might share a common ancestor, thus suggesting a broad hyaluronidase-like distribution among

  1. Cloning and characterization of a novel Athspr promoter specifically active in vascular tissue.

    PubMed

    Zhang, Liang; Yang, Tao; Li, Xiaoying; Hao, Hongyan; Xu, Shengtao; Cheng, Wei; Sun, Yingli; Wang, Chongying

    2014-05-01

    The vascular system--xylem, phloem and the cambium--is essential for water supply, nutrient transport, and physical support in higher plants. Although it is known that vascular-specific gene expression is regulated by cis-acting regulatory sequences in promoters, it is largely unknown how many regulatory elements exist and what their roles are in promoters. To understand the regulatory elements of vascular-specific promoters and their roles in vascular development, a T-DNA insertion mutant showing delayed growth and diminished resistance to environmental stress was isolated using promoter trap strategy. The novel gene, Arabidopsis thaliana heat shock protein-related (Athspr), was cloned from Arabidopsis ecotype C24. Strong GUS (β-glucuronidase) staining in the original promoter trap line was found in the vascular tissues of all organs in the mutant. The Athspr promoter was cloned and fused with GUS and eGFP (enhanced green fluorescent protein) reporter genes to verify its vascular-specific expression in Arabidopsis. Further histochemical analysis in transgenic plants demonstrated a similar GUS expression pattern in the vascular tissues. In addition, ATHSPR-eGFP driven by Athspr promoter was observed in vascular bundles of the transgenic seedling roots. Finally, comparative analysis with promoter motifs from 37 genes involved in vascular development revealed that Athspr and all other promoters active in vascular tissues contained regulatory elements responding to phytohormones, light, biotic and abiotic stresses, as well as those regulating tissue-specific expression. These results demonstrated that the Athspr promoter has a vascular tissue-specific activity and Athspr may have multiple functions in vascular development and resistance against various stresses. PMID:24675528

  2. Molecular cloning and characterization of multiple isoforms of the snowdrop (Galanthus nivalis L.) lectin.

    PubMed

    Van Damme, E J; De Clercq, N; Claessens, F; Hemschoote, K; Peeters, B; Peumans, W J

    1991-12-01

    Screening of a copy-DNA (cDNA) library constructed from RNA isolated from young developing ovaries of snowdrop (Galanthus nivalis) resulted in the isolation of five lectin clones which clearly differed from each other with regard to their nucleotide sequence and deduced amino-acid sequence. Sequence comparison between the coding regions of different lectin cDNAs revealed the highest homology between lectin clones LECGNA 3 and LECGNA 5, showing 96.4% and 93.6% similarity at the nucleotide level and at the deduced amino-acid level, respectively, whereas lectin clones LECGNA 1 and LECGNA 3 showed the lowest homology of 81.6% and 68.6% for the nucleotide sequence and the amino-acid sequence, respectively. Only very few lectin cDNA clones containing a polyadenylated tail could be isolated. Moreover all these cDNA clones were derived from isolectin 3 and showed some variability within the length of the 3' untranslated region. The major transcription initiation site was located 30 bases upstream from the AUG codon as could be deduced from primer-extension analysis. Taking into account the small 5' untranslated region of the lectin clones, the size of the lectin mRNA, which is approx. 780 nucleotides as determined by Northern blot analysis, is in good agreement with the length of the cDNA clones isolated. Besides the ovary tissue, both the leaf and the flower tissue were also shown to express the lectin mRNA in a flowering snowdrop plant.

  3. Molecular cloning of cecropin B responsive endonucleases in Yersinia ruckeri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were ...

  4. Molecular cloning of a human macrophage lectin specific for galactose

    SciTech Connect

    Cherayil, B.J.; Chairovitz, S.; Wong, C.; Pillai, S. Harvard Medical School, Boston )

    1990-09-01

    The murine Mac-2 protein is a galactose- and IgE-binding lectin secreted by inflammatory macrophages. The authors describe here the cloning an dcharacterization of cDNA representing the human homolog of Mac-2 (hMac-2). The amino acid sequence derived from the hMac-2 cDNA indicates that the protein is evolutionarily highly conserved, with 85% of its amino acid residues being similar to those in the murine homolog. This conservation is especially marked in the carboxyl-terminal lectin domain. The amino-terminal half of the protein is less conserved but still contains the repetitive proline-glycine-rich motif seen in the mouse protein. hMac-2 synthesized in vitro is recognized by the M3/38 monoclonal antibody to Mac-2 and binds to the desialylated glycoprotein asialofetuin and to laminin, a major component of basement membranes. These findings are discussed in the context of the potential functions of hMac-2.

  5. Molecular cloning of ADP-glucose pyrophosphorylase from cyanobacteria

    SciTech Connect

    Kakefuda, G.; Yeeyung Charng; Iglesias, A.; McIntosh, L. )

    1991-05-01

    Bacterial and higher plant ADP-glucose pyrophosphorylase differ in structure (homotetramer vs heterotetramer respectively) and allosteric activator and inhibitor. However, highly conserved regions can be identified when sequence comparisons are made between ADP-glucose pyrophosphorylases from diverse species. The fructose 1,6 bisphosphate binding site (activator site) in E. coli is highly conserved in all species for which ADP-glucose pyrophosphorylase has been sequenced. A second conserved region, which is labeled by 8-azido-ATP, is also highly conserved in bacteria and higher plants. In previously cloned ADP-glucose pyrophosphorylases the two conserved regions are separated by approximately 80 amino acids. The authors have used these conserved amino acid sequences to design degenerate oligonucleotide primers for polymerase chain reaction amplification (PCR) of part of the ADP-glucose pyrophosphorylase geae. A predicted 240 bp fragment is amplified in PCR reactions using Anabaena sp. PCC 7120 and Synechocystis sp. PCC 6803 genomic DNA as template. The deduced amino acid sequence from the 240 bp Anabaena fragment shares 75 and 76% identity to that of the rice endosperm and spinach leaf ADP-glucose pyrophosphorylases respectively. The Anabaena amino acid sequence shares 42% identity in amino acid sequence to the E. coli enzyme. At the nucleotide level there is 66% identity of the Anabaena sequence to rice endosperm ADP-glucose pyrophosphorylase and 54% to the E. coli gene. The PCR amplified fragments are being used to screen respective Anabaena and Synechocystis genomic gene libraries.

  6. Molecular Cloning of Tomato Plasma Membrane H+-ATPase 1

    PubMed Central

    Ewing, Nicholas N.; Wimmers, Larry E.; Meyer, David J.; Chetelat, Roger T.; Bennett, Alan B.

    1990-01-01

    Two cDNA clones (LHA1 and LHA2) from tomato (Lycopersicon esculentum) which likely encode isoforms of the plasma membrane H+-ATPase were isolated. The longest cDNA (3229 base pairs), LHA1, comprises an open reading frame that encodes a 956 amino acid, 105 kilodalton polypeptide with several potential transmembrane domains. In vitro transcription and translation of LHA1 yields a major translation product of approximately 100 kilodaltons that is immunoprecipitable with antiserum to the corn root plasma membrane H+-ATPase. LHA2 encodes a portion of a coding sequence that is 96% identical to LHA1, suggesting that LHA2 encodes an isoform of the H+-ATPase. Genomic DNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to a common set of six to eight restriction fragments at moderate stringency and to single distinct fragments at high stringency. LHA1 and LHA2 map to distinct sites on chromosomes three and six, respectively. RNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to 3.4 kilobase pair transcripts present in both leaves and roots, although the LHA2 transcript is relatively more abundant in leaves than in roots. These results indicate that in tomato as many as six to eight genes may encode the plasma membrane H+-ATPase, two of which are expressed at the level of mRNA in both roots and leaves. Images Figure 3 Figure 4 Figure 5 Figure 7 PMID:16667929

  7. Molecular cloning, expression, and enzymatic analysis of cathepsin X from starfish (Asterina pectinifera).

    PubMed

    Bak, Hye Jin; Kim, Moo-Sang; Kim, Na Young; Go, Hye-Jin; Han, Jin Woo; In Jo, Hyae; Ahn, Sang Jung; Park, Nam Gyu; Chung, Joon Ki; Lee, Hyung Ho

    2013-02-01

    Cathepsin X, also known as cathepsin Z, is referred to as a "lysosomal proteolytic enzyme" and a member of the peptidase C1 family, which is involved in various biological processes such as immune response, cell adhesion, and proliferation. In the present study, the cDNA of starfish (Asterina pectinifera), which is known to cause serious damage to commercial shellfish mariculture, cathepsin X (ApCtX) was isolated through the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) methods for the application to find a way to reduce/control starfish densities. The full-length of ApCtX gene was determined to consist of the 2,240 bp nucleotide sequence, which encoded for a preproprotein of 296 amino acids with a molecular mass of about 32.7 kDa. The tissue type expression of ApCtX was determined in various tissues of A. pectinifera and was shown most abundantly in the liver. The cDNA encoding pro-mature enzyme of ApCtX was expressed in Escherichia coli BL21 (DE3) using the pGEX-4T-1 expression vector. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC. The optimal pH for the protease activity was 6.5. The enzymatic activity of proApCtX was reduced by antipain, NEM, EDTA, EGTA, and 1,10-phenanthroline, and the proApCtX enzyme was significantly inhibited by CuSO₄, HgCl₂, CoCl₂, and SDS whereas Triton X-100 and Brij 35 might have potentially acted as an activator. Here, we demonstrated for the first time that the structural features and enzymatic characteristics of Echinoderms cathepsin X are similar to those of the other mammalian and piscine cathepsin X except its pH optimum, and the results of tissue-specific expression might explain their importance in food digestion by hepatic cecain starfish.

  8. Molecular Characterization of Infectious Clones of the Minute Virus of Canines Reveals Unique Features of Bocaviruses▿

    PubMed Central

    Sun, Yuning; Chen, Aaron Yun; Cheng, Fang; Guan, Wuxiang; Johnson, F. Brent; Qiu, Jianming

    2009-01-01

    Minute virus of canines (MVC) is a member of the genus Bocavirus in the family Parvoviridae. We have molecularly cloned and sequenced the 5′- and 3′-terminal palindromes of MVC. The MVC genome, 5,404 nucleotides (nt) in length, shared an identity of 52.6% and 52.1% with that of human bocavirus and bovine parvovirus, respectively. It had distinct palindromic hairpins of 183 nt and 198 nt at the left-end and right-end termini of the genome, respectively. The left-end terminus was also found in two alternative orientations (flip or flop). Both termini shared extensive similarities with those of bovine parvovirus. Four full-length molecular clones constructed with different orientations of the left-end terminus proved to be infectious in Walter Reed canine cell/3873D (WRD) canine cells. Both MVC infection and transfection of the infectious clone in WRD cells revealed an identical RNA transcription profile that was similar to that of bovine parvovirus. Mutagenesis of the infectious clone demonstrated that the middle open reading frame encodes the NP1 protein. This protein, unique to the genus Bocavirus, was essential for MVC DNA replication. Moreover, the phospholipase A2 motif in the VP1 unique region was also critical for MVC infection. Thus, our studies revealed important information about the genus Bocavirus that may eventually help us to clone the human bocavirus and study its pathogenesis. PMID:19211770

  9. Molecular Cloning and Characterization of Growth Factor Receptor Bound-Protein in Clonorchis sinensis

    PubMed Central

    Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong

    2014-01-01

    Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892

  10. Molecular cloning and functional characterization of murine toll‑like receptor 8.

    PubMed

    Li, Tingting; He, Xiaobing; Jia, Huaijie; Chen, Guohua; Zeng, Shuang; Fang, Yongxiang; Jin, Qiwang; Jing, Zhizhong

    2016-02-01

    Toll-like receptors (TLRs) are a large family of germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns and evoke the relevant innate immune responses. TLR8 is a member of several endosome nucleic acid-sensing TLRs; however little attention has been paid to murine TLR8 (mTLR8) compared with other endosome nucleic acid-sensing TLRs. In the present study, mTLR8 was cloned using reverse transcription-polymerase chain reaction from murine peripheral blood mononuclear cells and its function in regulating innate immune response was characterized. The open reading frame of mTLR8 consists of 3,099 bps and encodes 1,032 amino acids. It contains typical leucine-rich repeats, a transmembrane domain and a Toll/interleukin-1 receptor domain, and it shares a high level of identity with other mammalian species. The expression of mTLR8 has been widely observed in different tissues, and higher expression levels of mTLR8 have mainly been detected in the heart, spleen and lung. Overexpression of mTLR8 is required for the activation of transcription factor nuclear factor-κB and the production of tumor necrosis factor-α. However, mTLR8 is not able to activate interferon regulatory factor 3 or activator protein 1, nor can it induce interferon-α in HEK293T cells. These results indicate that mTLR8, as an important PRR, is indeed functional and is vital role in the activation of innate immune responses. This study may aid in determining the molecular basis of the interactions between mTLR8 and pathogens. PMID:26676274

  11. Molecular cloning and developmental expression of plakophilin 2 in zebrafish

    SciTech Connect

    Moriarty, Miriam A.; Martin, Eva D.; Byrnes, Lucy; Grealy, Maura

    2008-02-29

    Armadillo proteins are involved in providing strength and support to cells and tissues, nuclear transport, and transcriptional activation. In this report, we describe the identification and characterisation of the cDNA of the desmosomal armadillo protein plakophilin 2 in zebrafish. The 2448 bp coding sequence encodes a predicted 815 amino acid protein, with nine armadillo repeats characteristic of the p120-catenin subfamily. It shares conserved N-glycosylation, myristoylation, and glycogen synthase kinase 3, casein kinase 2, and protein kinase C phosphorylation sites with mammalian armadillo proteins including plakoglobin and {beta}-catenin. Semi-quantitative reverse transcription polymerase chain reaction and whole mount in situ hybridisation show that it is expressed both maternally and zygotically. It is ubiquitously expressed during blastula stages but becomes restricted to epidermal and cardiac tissue during gastrulation. These results provide evidence that zebrafish plakophilin 2 is developmentally regulated with potential roles in cell adhesion, signalling, and cardiac and skin development.

  12. Differential Cell Tropism of Feline Immunodeficiency Virus Molecular Clones In Vivo

    PubMed Central

    Dean, Gregg A.; Himathongkham, Sunee; Sparger, Ellen E.

    1999-01-01

    Independent studies have demonstrated different cell tropisms for molecular clones of feline immunodeficiency virus (FIV). In this report, we examined three clones, FIV-pF34, FIV-14, and FIV-pPPR, for replication in Crandell feline kidney (CrFK) cells, feline peripheral blood mononuclear cells (PBMC), and feline macrophage cultures. Importantly, cell tropism for these three clones was also examined in vivo. FIV-pF34 replication was efficient in CrFK cells but severely restricted in PBMC, whereas replication of FIV-pPPR was vigorous in PBMC but severely restricted in CrFK cells. FIV-14 replication was productive in both CrFK cells and PBMC. Interestingly, all three molecular clones replicated with similar efficiencies in primary feline monocyte-derived macrophages. In vivo, FIV-pF34 proved least efficient for establishing persistent infection, and proviral DNA when detectable, was localized predominately to nonlymphoid cell populations (macrophages). FIV-pPPR proved most efficient for induction of a persistent viremia in vivo, and proviral DNA was localized predominately in CD4+ and CD8+ lymphocyte subsets. FIV-14 inoculation of cats resulted in an infection characterized by seroconversion and localization of proviral DNA in CD4+ lymphocytes only. Results of this study on diverse FIV molecular clones revealed that in vitro replication efficiency of an FIV isolate in PBMC directly correlated with replication efficiency in vivo, whereas proficiency for replication in macrophages in vitro was not predictive for replication potential in vivo. Also, infection of both CD4+ and CD8+ lymphocyte subsets was associated with higher virus load in vivo. Results of the studies on these three FIV clones, which exhibited differential cell tropism, indicated a correlation between in vitro and in vivo cell tropism and virus replication. PMID:10074104

  13. Molecular cloning and expression analysis of mulberry MAPK gene family.

    PubMed

    Wei, Congjin; Liu, Xueqin; Long, Dingpei; Guo, Qing; Fang, Yuan; Bian, Chenkai; Zhang, Dayan; Zeng, Qiwei; Xiang, Zhonghuai; Zhao, Aichun

    2014-04-01

    Mitogen-activated protein kinase (MAPK) cascades play an important role in regulating various biotic and abiotic stresses in plants. Although MAPKs have been identified and characterized in a few model plants, there is little information available for mulberry Morus sp. L., one of the most ecologically and economically important perennial trees. This study identified 47 mulberry Morus notabilis MAPK (MnMAPK) family genes: 32 MnMAPKKK, five MnMAPKK and ten MnMAPK genes, and cloned ten MnMAPK cDNA genes based on a genome-wide analysis of the morus genome database. Comparative analysis with MAPK gene families from other plants suggested that MnMAPKs could be divided into five subfamilies (groups A, B, C, D and E) and they could have similar functions in response to abiotic and biotic stresses. MnMAPK gene expression analysis of different stresses (high/low temperature, salt and drought) and signal molecules (ABA, SA, H2O2 and methyl jasmonate (MeJA)) revealed that all ten MnMAPK genes responded to high/low temperature, salt and drought stresses, and that nine of the ten MnMAPKs (MnMAPK7 excepted) could be induced by ABA, SA, H2O2 and MeJA, which suggested that MnMAPKs may play pivotal roles in signal transduction pathways. Our results indicated that almost all of the MnMAPKs may be involved in environmental stress and defense responses, which provides the basis for further characterization of the physiological functions of MnMAPKs.

  14. Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

    PubMed Central

    Yoshikane, Yu; Yokochi, Nana; Ohnishi, Kouhei; Hayashi, Hideyuki; Yagi, Toshiharu

    2006-01-01

    Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed. PMID:16545075

  15. Molecular cloning and analysis of the Catsper1 gene promoter.

    PubMed

    Mata-Rocha, Minerva; Alvarado-Cuevas, Edith; Hernández-Sánchez, Javier; Cerecedo, Doris; Felix, Ricardo; Hernández-Reyes, Adriana; Tesoro-Cruz, Emiliano; Oviedo, Norma

    2013-05-01

    CatSper channels are essential for hyperactivity of sperm flagellum, progesterone-mediated chemotaxis and oocyte fertilization. Catsper genes are exclusively expressed in the testis during spermatogenesis, but the function and regulation of the corresponding promoter regions are unknown. Here, we report the cloning and characterization of the promoter regions in the human and murine Catsper1 genes. These promoter regions were identified and isolated from genomic DNA, and transcriptional activities were tested in vitro after transfection into human embryonic kidney 293, mouse Sertoli cells 1 and GC-1spg cell lines as well as by injecting plasmids directly into mouse testes. Although the human and murine Catsper1 promoters lacked a TATA box, a well-conserved CRE site was identified. Both sequences may be considered as TATAless promoters because their transcriptional activity was not affected after deletion of TATA box-like sites. Several transcription initiation sites were revealed by RNA ligase-mediated rapid amplification of the cDNA 5'-ends. We also found that the immediate upstream region and the first exon in the human CATSPER1 gene negatively regulate transcriptional activity. In the murine Catsper1 promoter, binding sites for transcription factors SRY, SOX9 and CREB were protected by the presence of nuclear testis proteins in DNAse degradation assays. Likewise, the mouse Catsper1 promoter exhibited transcriptional activity in both orientations and displayed significant expression levels in mouse testis in vivo, whereas the suppression of transcription signals in the promoter resulted in low expression levels. This study, thus, represents the first identification of the transcriptional control regions in the genes encoding the human and murine CatSper channels.

  16. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time.

  17. [Cloning and tissue expression of 4-coumarate coenzyme A ligase gene in Angelica sinensis].

    PubMed

    Wen, Sui-chao; Wang, Yin-quan; Luo, Jun; Xia, Qi; Fan, Qin; Li, Shu-nan; Wang, Zhen-heng

    2015-12-01

    4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis. PMID:27245029

  18. Construction and characterization of an infectious molecular clone of Koala retrovirus.

    PubMed

    Shojima, Takayuki; Hoshino, Shigeki; Abe, Masumi; Yasuda, Jiro; Shogen, Hiroko; Kobayashi, Takeshi; Miyazawa, Takayuki

    2013-05-01

    Koala retrovirus (KoRV) is a gammaretrovirus that is currently endogenizing into koalas. Studies on KoRV infection have been hampered by the lack of a replication-competent molecular clone. In this study, we constructed an infectious molecular clone, termed plasmid pKoRV522, of a KoRV isolate (strain Aki) from a koala reared in a Japanese zoo. The virus KoRV522, derived from pKoRV522, grew efficiently in human embryonic kidney (HEK293T) cells, attaining 10(6) focus-forming units/ml. Several mutations in the Gag (L domain) and Env regions reported to be involved in reduction in viral infection/production in vitro are found in pKoRV522, yet KoRV522 replicated well, suggesting that any effects of these mutations are limited. Indeed, a reporter virus pseudotyped with pKoRV522 Env was found to infect human, feline, and mink cell lines efficiently. Analyses of KoRV L-domain mutants showed that an additional PPXY sequence, PPPY, in Gag plays a critical role in KoRV budding. Altogether, our results demonstrate the construction and characterization of the first infectious molecular clone of KoRV. The infectious clone reported here will be useful for elucidating the mechanism of endogenization of the virus in koalas and screening for antiretroviral drugs for KoRV-infected koalas.

  19. Tissue engineering, stem cells and cloning: current concepts and changing trends.

    PubMed

    Atala, Anthony

    2005-07-01

    Organ damage or loss can occur from congenital disorders, cancer, trauma, infection, inflammation, iatrogenic injuries or other conditions and often necessitates reconstruction or replacement. Replacement may take the form of organ transplant. At present, there is a severe shortage of donor organs that is worsening with the aging of the population. Tissue engineering follows the principles of cell transplantation, materials science and engineering towards the development of biological substitutes that can restore and maintain normal tissue function. Therapeutic cloning involves the introduction of a nucleus from a donor cell into an enucleated oocyte to generate embryonic stem cell lines whose genetic material is identical to that of its source. These autologous stem cells have the potential to become almost any type of cell in the adult body, and thus would be useful in tissue and organ replacement applications. This paper reviews recent advances in stem cell research and regenerative medicine, and describes the clinical applications of these technologies as novel therapies for tissue or organ loss.

  20. Cloning and analysis of differentially expressed ESTs in swine muscle tissue.

    PubMed

    Li, Chongsheng; Chen, Yaosheng; Wang, Chong; Li, Jiaqi

    2006-08-01

    The obvious difference in muscle growth and meat quality traits exists between Chinese indigenous pig and exotic pigs. In order to study the reason of these phenotypic differences and search the potential gene related to growth and meat quality traits, silver-stained mRNA differential display technique was used to detect the difference with mRNA of loin-eye muscle tissue from maturity pigs of Lantang in Guangdong Province and Large Yorkshire. One of the newly discovered expressed sequence tag (ESTsp3) was analyzed by using bioinformatic technique. The results showed: (1) nearly 2000 cDNA fragments were detected with 30 primer pairs, and 6 differentially expressed ESTs in the loin-eye muscle tissues from the two breeds were isolated and obtained. The differential fragments were cloned and sequenced. The all sequences were recorded in the GenBank. (2) The 786 bp fragment of ESTsp3 was obtained with in silico elongation system, the ORF analysis revealed that it existed as an 83 aa complete open reading frame, and the elongation sequences were verified by RT-PCR. The analysis of in silico expression profile showed that ESTsp3 is expressed in various growth stages and in most tissues and organs, such as soft tissue, skin, skeletal muscle and kidney, but with variant expression quantity.

  1. Molecular cloning and mRNA expression of cyclophilin A gene in black tiger shrimp (Penaeus monodon).

    PubMed

    Qiu, Lihua; Jiang, Shigui; Huang, Jianhua; Wang, Weifang; Zhu, Caiyan; Su, Tianfeng

    2009-01-01

    The techniques of homology cloning and anchored PCR were used to clone the cyclophilin A (CypA) gene from black tiger shrimp (Penaeus monodon). The full-length cDNA of black tiger shrimp CypA (btsCypA) contained a 5' untranslated region (UTR) of 81 bp, an ORF (open reading frame) of 495 bp encoding a polypeptide of 164 amino acids with an estimated molecular mass of 17.68 kDa and a 3' UTR of 308 bp. The predicted amino acid sequence of btsCypA shared high identity with CypA in other organisms. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of btsCypA in different tissues and the temporal expression of btsCypA in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of btsCypA was detected in the tissues of hepatopancreas and blood. The expression of btsCypA in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that btsCypA was a constitutive and inducible expressed protein and could be induced by LPS.

  2. Molecular cloning and expression analysis of a F-type lectin gene from Japanese sea perch (Lateolabrax japonicus).

    PubMed

    Qiu, Lihua; Lin, Liansheng; Yang, Keng; Zhang, Hanhua; Li, Jianzhu; Zou, Falin; Jiang, Shigui

    2011-08-01

    The techniques of homology cloning and anchored PCR were used to clone the fucose-binding lectin (F-type lectin) gene from Japanese sea perch (Lateolabrax Japonicus). The full-length cDNA of sea perch F-lectin (JspFL) contained a 5' untranslated region (UTR) of 39 bp, an ORF of 933 bp encoding a polypeptide of 310 amino acids with an estimated molecular mass of 10.82 kDa and a 3' UTR of 332 bp. The searches for nucleotides and protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of JspFL was homological to the Fucose-binding lectin in other fish species. In the JspFL deduced amino acid sequence, two tandem domains that exhibit the eel carbohydrate-recognition sequence motif were found. The temporal expressions of gene in the different tissues were measured by real-time PCR. And the mRNA expressions of the gene were constitutively expressed in tissues including spleen, head-kidney, liver, gill, and heart. The JspFL expression in spleen was different during the stimulated time point, 2 h later the expression level became up-regulated, and 6 h later the expression level became down-regulated. The result indicated that JspFL was constitutive and inducible expressed and could play a critical role in the host-pathogen interaction.

  3. Purification, characterization and cloning of tensilin, the collagen-fibril binding and tissue-stiffening factor from Cucumaria frondosa dermis.

    PubMed

    Tipper, Jennifer P; Lyons-Levy, Gillian; Atkinson, Mark A L; Trotter, John A

    2002-12-01

    The inner dermis of the sea cucumber, Cucumaria frondosa, is a mutable collagenous tissue characterized by rapid and reversible changes in its mechanical properties regulated by one or more protein effectors that are released from neurosecretory cells. One such effector, tensilin, is a collagen-fibril binding protein, named for its ability to induce dermis stiffening. Tensilin was purified using an affinity column constructed from C. frondosa collagen-fibrils. The protein migrates as a single band on SDS-PAGE (Mr approximately 33 kDa) and has an isoelectric point of 5.8. Equilibrium sedimentation experiments suggest a molecular mass of approximately 28.5-29.4 kDa. Carbohydrate analysis of tensilin revealed no measurable sugar content. The molar amount of tensilin was determined to be 0.38% that of collagen and 47% that of stiparin, a constitutive matrix glycoprotein. A full-length cDNA clone for tensilin was obtained from a C. frondosa inner dermis cDNA expression library. Predicted properties derived from the deduced peptide sequence were in agreement with those of the native protein. A noted feature of tensilin's deduced peptide sequence, particularly in its N-terminal domain, is its homology to tissue inhibitor of metalloproteinases. Tensilin's C-terminal tail has no known homology to other proteins but contains a putative collagen-fibril binding site.

  4. Development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia virus.

    PubMed

    Cook, R F; Leroux, C; Cook, S J; Berger, S L; Lichtenstein, D L; Ghabrial, N N; Montelaro, R C; Issel, C J

    1998-02-01

    An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon of rev), and the 3' long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV(PV3.3), and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAV(PV3.3#3) (redesignated EIAV(UK)), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6 degrees C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3' fragment of EIAV(UK) differed from the consensus sequence of EIAV(PV) by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV(PV) consensus sequence was observed in the hypervariable region of the LTR. However, EIAV(UK) was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAV(PV) strain into the infectious nonpathogenic

  5. Molecular cloning and characterization of the light-harvesting chlorophyll a/b gene from the pigeon pea (Cajanus cajan).

    PubMed

    Qiao, Guang; Wen, Xiao-Peng; Zhang, Ting

    2015-12-01

    Light-harvesting chlorophyll a/b-binding proteins (LHCB) have been implicated in the stress response. In this study, a gene encoding LHCB in the pigeon pea was cloned and characterized. Based on the sequence of a previously obtained 327 bp Est, a full-length 793 bp cDNA was cloned using the rapid amplification of cDNA ends (RACE) method. It was designated CcLHCB1 and encoded a 262 amino acid protein. The calculated molecular weight of the CcLHCB1 protein was 27.89 kDa, and the theoretical isoelectric point was 5.29. Homology search and sequence multi-alignment demonstrated that the CcLHCB1 protein sequence shared a high identity with LHCB from other plants. Bioinformatics analysis revealed that CcLHCB1 was a hydrophobic protein with three transmembrane domains. By fluorescent quantitative real-time polymerase chain reaction (PCR), CcLHCB1 mRNA transcripts were detectable in different tissues (leaf, stem, and root), with the highest level found in the leaf. The expression of CcLHCB1 mRNA in the leaves was up-regulated by drought stimulation and AM inoculation. Our results provide the basis for a better understanding of the molecular organization of LCHB and might be useful for understanding the interaction between plants and microbes in the future. PMID:26329890

  6. Molecular cloning and characterization of the light-harvesting chlorophyll a/b gene from the pigeon pea (Cajanus cajan).

    PubMed

    Qiao, Guang; Wen, Xiao-Peng; Zhang, Ting

    2015-12-01

    Light-harvesting chlorophyll a/b-binding proteins (LHCB) have been implicated in the stress response. In this study, a gene encoding LHCB in the pigeon pea was cloned and characterized. Based on the sequence of a previously obtained 327 bp Est, a full-length 793 bp cDNA was cloned using the rapid amplification of cDNA ends (RACE) method. It was designated CcLHCB1 and encoded a 262 amino acid protein. The calculated molecular weight of the CcLHCB1 protein was 27.89 kDa, and the theoretical isoelectric point was 5.29. Homology search and sequence multi-alignment demonstrated that the CcLHCB1 protein sequence shared a high identity with LHCB from other plants. Bioinformatics analysis revealed that CcLHCB1 was a hydrophobic protein with three transmembrane domains. By fluorescent quantitative real-time polymerase chain reaction (PCR), CcLHCB1 mRNA transcripts were detectable in different tissues (leaf, stem, and root), with the highest level found in the leaf. The expression of CcLHCB1 mRNA in the leaves was up-regulated by drought stimulation and AM inoculation. Our results provide the basis for a better understanding of the molecular organization of LCHB and might be useful for understanding the interaction between plants and microbes in the future.

  7. [Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics].

    PubMed

    Camacho, Daría; Reyes, Jesús; Franco, Leticia; Comach, Guillermo; Ferrer, Elizabeth

    2016-06-01

    The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription- polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5'UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5'UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous. PMID:27656926

  8. [Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics].

    PubMed

    Camacho, Daría; Reyes, Jesús; Franco, Leticia; Comach, Guillermo; Ferrer, Elizabeth

    2016-06-01

    The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription- polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5'UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5'UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous.

  9. Molecular Cloning and Characterization of a Paramyosin from Clonorchis sinensis

    PubMed Central

    Park, Tae-Joon; Kang, Jung-Mi; Na, Byoung-Kuk

    2009-01-01

    Paramyosin is a myofibrillar protein present in helminth parasites and plays multifunctional roles in host-parasite interactions. In this study, we identified the gene encoding paramyosin of Clonorchis sinensis (CsPmy) and characterized biochemical and immunological properties of its recombinant protein. CsPmy showed a high level of sequence identity with paramyosin from other helminth parasites. Recombinant CsPmy (rCsPmy) expressed in bacteria had an approximate molecular weight of 100 kDa and bound both human collagen and complement 9. The protein was constitutively expressed in various developmental stages of the parasite. Imunofluorescence analysis revealed that CsPmy was mainly localized in the tegument, subtegumental muscles, and the muscle layer surrounding the intestine of the parasite. The rCsPmy showed high levels of positive reactions (74.6%, 56/75) against sera from patients with clonorchiasis. Immunization of experimental rats with rCsPmy evoked high levels of IgG production. These results collectively suggest that CsPmy is a multifunctional protein that not only contributes to the muscle layer structure but also to non-muscular functions in host-parasite interactions. Successful induction of host IgG production also suggests that CsPmy can be applied as a diagnostic antigen and/or vaccine candidate for clonorchiasis. PMID:19967083

  10. Molecular cloning and characterization of crustin from mud crab Scylla paramamosain.

    PubMed

    Imjongjirak, Chanprapa; Amparyup, Piti; Tassanakajon, Anchalee; Sittipraneed, Siriporn

    2009-05-01

    Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain. PMID:18425600

  11. Molecular cloning of rhamnose-binding lectin gene and its promoter region from snakehead Channa argus.

    PubMed

    Jia, W Z; Shang, N; Guo, Q L

    2010-09-01

    Lectins are sugar-binding proteins that mediate pathogen recognition and cell-cell interactions. A rhamnose-binding lectin (RBL) gene and its promoter region have been cloned and characterized from snakehead Channa argus. From the transcription initiation site, snakehead rhamnose-binding lectin (SHL) gene extends 2,382 bp to the end of the 3' untranslated region (UTR), and contains nine exons and eight introns. The open reading frame (ORF) of the SHL transcript has 675 bp which encodes 224 amino acids. The molecular structure of SHL is composed of two tandem repeat carbohydrate recognition domains (CRD) with 35% internal identity. Analysis of the gene organization of SHL indicates that the ancestral gene of RBL may diverge and evolve by exon shuffling and gene duplication, producing new forms to play their own roles in various organisms. The characteristics of SHL gene 5' flanking region are the presence of consensus nuclear factor of interleukin 6 (NF-IL6) and IFN-gamma activation (GAS) sites. The results provide indirect evidence that up-regulation of SHL expression may be induced in response to inflammatory stimuli, such as lipopolysaccharide (LPS), interleukin 6 (IL-6), and interferon gamma (IFN-gamma). The transcript of SHL mRNA was expressed in the head kidney, posterior kidney, spleen, liver, intestine, heart, muscle, and ovary. No tissue-specific expressive pattern is different from reported STLs, WCLs, and PFLs, suggesting that different types of RBLs exist in species-specific fish that have evolved and adapted to their surroundings.

  12. Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

    NASA Astrophysics Data System (ADS)

    Wang, Qing; Ning, Xuanxuan; Pei, Dong; Zhao, Jianmin; You, Liping; Wang, Chunyan; Wu, Huifeng

    2013-05-01

    Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis. In this study, two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplifi cation of cDNA ends-polymerase chain reaction (RACE-PCR). The two cDNAs were named MgTrx1 and MgTrx2, respectively. The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa, respectively. Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys. In addition, MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria. Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined. The MgTrx1 transcript was most abundant in hemocytes and gills, whereas the MgTrx2 transcript was most abundant in gonad, hepatopancreas, gill and hemocytes. Following Vibrio anguillarum challenge, the expression of MgTrx1 was up-regulated and reached its peak, at a value 10-fold the initial value, at 24 h. Subsequently, expression returned back to the original level. In contrast, the expression level of MgTrx2 was down-regulated following bacterial stimulation, with one fi fth of the control level evident at 12 h post challenge. These results suggest that MgTrx1 and MgTrx2 may play important roles in the response of M. galloprovincialis to bacterial challenge.

  13. Molecular Portrait of the Normal Human Breast Tissue and Its Influence on Breast Carcinogenesis

    PubMed Central

    Margan, Madalin Marius; Jitariu, Andreea Adriana; Nica, Cristian; Raica, Marius

    2016-01-01

    Normal human breast tissue consists of epithelial and nonepithelial cells with different molecular profiles and differentiation grades. This molecular heterogeneity is known to yield abnormal clones that may contribute to the development of breast carcinomas. Stem cells that are found in developing and mature breast tissue are either positive or negative for cytokeratin 19 depending on their subtype. These cells are able to generate carcinogenesis along with mature cells. However, scientific data remains controversial regarding the monoclonal or polyclonal origin of breast carcinomas. The majority of breast carcinomas originate from epithelial cells that normally express BRCA1. The consecutive loss of the BRCA1 gene leads to various abnormalities in epithelial cells. Normal breast epithelial cells also express hypoxia inducible factor (HIF) 1α and HIF-2α that are associated with a high metastatic rate and a poor prognosis for malignant lesions. The nuclear expression of estrogen receptor (ER) and progesterone receptor (PR) in normal human breast tissue is maintained in malignant tissue as well. Several controversies regarding the ability of ER and PR status to predict breast cancer outcome remain. Both ER and PR act as modulators of cell activity in normal human breast tissue. Ki-67 positivity is strongly correlated with tumor grade although its specific role in applied therapy requires further studies. Human epidermal growth factor receptor 2 (HER2) oncoprotein is less expressed in normal human breast specimens but is highly expressed in certain malignant lesions of the breast. Unlike HER2, epidermal growth factor receptor expression is similar in both normal and malignant tissues. Molecular heterogeneity is not only found in breast carcinomas but also in normal breast tissue. Therefore, the molecular mapping of normal human breast tissue might represent a key research area to fully elucidate the mechanisms of breast carcinogenesis. PMID:27382385

  14. Molecular Portrait of the Normal Human Breast Tissue and Its Influence on Breast Carcinogenesis.

    PubMed

    Margan, Madalin Marius; Jitariu, Andreea Adriana; Cimpean, Anca Maria; Nica, Cristian; Raica, Marius

    2016-06-01

    Normal human breast tissue consists of epithelial and nonepithelial cells with different molecular profiles and differentiation grades. This molecular heterogeneity is known to yield abnormal clones that may contribute to the development of breast carcinomas. Stem cells that are found in developing and mature breast tissue are either positive or negative for cytokeratin 19 depending on their subtype. These cells are able to generate carcinogenesis along with mature cells. However, scientific data remains controversial regarding the monoclonal or polyclonal origin of breast carcinomas. The majority of breast carcinomas originate from epithelial cells that normally express BRCA1. The consecutive loss of the BRCA1 gene leads to various abnormalities in epithelial cells. Normal breast epithelial cells also express hypoxia inducible factor (HIF) 1α and HIF-2α that are associated with a high metastatic rate and a poor prognosis for malignant lesions. The nuclear expression of estrogen receptor (ER) and progesterone receptor (PR) in normal human breast tissue is maintained in malignant tissue as well. Several controversies regarding the ability of ER and PR status to predict breast cancer outcome remain. Both ER and PR act as modulators of cell activity in normal human breast tissue. Ki-67 positivity is strongly correlated with tumor grade although its specific role in applied therapy requires further studies. Human epidermal growth factor receptor 2 (HER2) oncoprotein is less expressed in normal human breast specimens but is highly expressed in certain malignant lesions of the breast. Unlike HER2, epidermal growth factor receptor expression is similar in both normal and malignant tissues. Molecular heterogeneity is not only found in breast carcinomas but also in normal breast tissue. Therefore, the molecular mapping of normal human breast tissue might represent a key research area to fully elucidate the mechanisms of breast carcinogenesis. PMID:27382385

  15. Influence of molecular size on tissue distribution of antibody fragments

    PubMed Central

    Li, Zhe; Krippendorff, Ben-Fillippo; Sharma, Sharad; Walz, Antje C.; Lavé, Thierry; Shah, Dhaval K.

    2016-01-01

    Biodistribution coefficients (BC) allow estimation of the tissue concentrations of proteins based on the plasma pharmacokinetics. We have previously established the BC values for monoclonal antibodies. Here, this concept is extended by development of a relationship between protein size and BC values. The relationship was built by deriving the BC values for various antibody fragments of known molecular weight from published biodistribution studies. We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. The relationship was validated by a priori predicting BC values of 4 antibody fragments that were not used in building the relationship. The relationship was also used to derive BC50 values for all the tissues, which is the molecular weight increase that would result in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35 kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues. PMID:26496429

  16. Molecular cloning and characterization of presenilin gene in Bombyx mori.

    PubMed

    Zheng, Zeng-Zhang; Chao, Meng-Ling; Fan, Zong-Biao; Zhao, Yi-Jiao; Song, Hong-Sheng

    2015-10-01

    Presenilin (PS), the catalytic core of the γ-secretase complex, is considered to be a causative protein of the early‑onset familial form of Alzheimer's disease. Aging is a risk factor for Alzheimer's disease and a number of genetic studies have utilized Bombyx mori (B. mori) as a model, making it possible to use B. mori to investigate Alzheimer's disease. However, the homologous gene of human PS in B. mori has remained to be elucidated. In the present study, the PS homologue gene in B. mori was identified and characterized, and six B. mori presenilin (BmPS) mRNA transcripts were generated by selecting multiple transcription start sites and/or alternative splice sites. The longest mRNA of BmPS (termed BmPS1) contains a 153 nt 5' untranslated region (UTR), a 1,440 nt open reading frame and a 1,063 nt 3' UTR. The predicted protein of BmPS1 consists of 479 amino acid residues and has two highly‑conserved aspartate residues, which form the catalytic core of aspartic proteases. It exhibits a sequence identity of ~44 and 51% with homologues in Homo sapiens and Drosophila melanogaster, respectively. However, the amino acid sequence of the BmPS loop region does not completely match between the two B. mori strains R13Q and Dazao. Genomic analysis revealed that B. mori had a single copy of the BmPS gene, which was composed of 14 exons. A total of four isoforms of BmPS (BmPS‑A, ‑B, ‑C and ‑D) owing to multiple transcriptional start sites and alternative splice sites were identified. The alternative splicing events occurring in the loop region improved the diversity of the BmPS protein and were detectable in all tissues, as determined using reverse transcription quantitative polymerase chain reaction (RT‑qPCR). Furthermore, the expression levels of BmPS in the brain at different developmental stages were detected using RT‑qPCR, and significantly higher expression levels of BmPS were found in the adult stage compared with those in the larval and pupal stages

  17. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    SciTech Connect

    Deymier, Martin J.; Claiborne, Daniel T.; Ende, Zachary; Ratner, Hannah K.; Kilembe, William; Hunter, Eric

    2014-11-15

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

  18. Molecular cloning and expression of the O4 polysaccharide gene cluster from Escherichia coli.

    PubMed

    Haraguchi, G E; Hull, R A; Krallmann-Wenzel, U; Hull, S I

    1989-02-01

    The Escherichia coli O4 serotype is common among isolates from urinary tract infections. The genes responsible for the biosynthesis of the O4 polysaccharide in a human uropathogenic E. coli were cloned and expressed in E. coli K-12. The recombinant plasmid pGH60, which conferred the O4 phenotype, encoded eight proteins with apparent molecular weights of 39, 36.5, 35, 32.8, 26, 24, 20.7 and 13 kDa.

  19. Immersing Undergraduate Students in the Research Experience: A Practical Laboratory Module on Molecular Cloning of Microbial Genes

    ERIC Educational Resources Information Center

    Wang, Jack T. H.; Schembri, Mark A.; Ramakrishna, Mathitha; Sagulenko, Evgeny; Fuerst, John A.

    2012-01-01

    Molecular cloning skills are an essential component of biological research, yet students often do not receive this training during their undergraduate studies. This can be attributed to the complexities of the cloning process, which may require many weeks of progressive design and experimentation. To address this issue, we incorporated an…

  20. A molecularly cloned, pathogenic, neutralization-resistant simian immunodeficiency virus, SIVsmE543-3.

    PubMed Central

    Hirsch, V; Adger-Johnson, D; Campbell, B; Goldstein, S; Brown, C; Elkins, W R; Montefiori, D C

    1997-01-01

    An infectious molecular clone of simian immunodeficiency virus SIVsm was derived from a biological isolate obtained late in disease from an immunodeficient rhesus macaque (E543) with SIV-induced encephalitis. The molecularly cloned virus, SIVsmE543-3, replicated well in macaque peripheral blood mononuclear cells and monocyte-derived macrophages and resisted neutralization by heterologous sera which broadly neutralized genetically diverse SIV variants in vitro. SIVsmE543-3 was infectious and induced AIDS when inoculated intravenously into pig-tailed macaques (Macaca nemestrina). Two of four infected macaques developed no measurable SIV-specific antibody and succumbed to a wasting syndrome and SIV-induced meningoencephalitis by 14 and 33 weeks postinfection. The other two macaques developed antibodies reactive in Western blot and virus neutralization assays. One macaque was sacrificed at 1 year postinoculation, and the survivor has evidence of immunodeficiency, characterized by persistently low CD4 lymphocyte subsets in the peripheral blood. Plasma samples from these latter animals neutralized SIVsmE543-3 but with much lower efficiency than neutralization of other related SIV strains, confirming the difficulty by which this molecularly cloned virus is neutralized in vitro. SIVsmE543-3 will provide a valuable reagent for studying SIV-induced encephalitis, mapping determinants of neutralization, and determining the in vivo significance of resistance to neutralization in vitro. PMID:8995688

  1. Molecular cloning, functional expression and pharmacological characterization of a mouse melanocortin receptor gene.

    PubMed Central

    Desarnaud, F; Labbe, O; Eggerickx, D; Vassart, G; Parmentier, M

    1994-01-01

    We describe the cloning of the mouse HGMP01A gene that encodes a melanocortin receptor functionally distinct from the adrenal cortex corticotropin (adrenocorticotrophic hormone; ACTH) receptor and the melanocyte-stimulating hormone (MSH) receptor expressed in melanoma. The gene encodes a protein of 323 amino acids with a calculated molecular mass of 35,800 Da, displaying potential sites for N-linked glycosylation and phosphorylation by protein kinase C. An RNAase protection assay detected weak expression in the brain, but not in adrenal gland, skin, or any of the other tissues tested. Stable CHO cell lines expressing over 100,000 receptors per cell were generated. The recombinant receptor binds iodinated [Nle4,D-Phe7]alpha-MSH (NDP-MSH) with an apparent Kd of 700 pM. Displacement of the ligand by a variety of pro-opiomelanocortin-derived peptides revealed a pharmacological profile distinct from that of the classical ACTH and MSH receptors. NDP-MSH was the most powerful competitor (IC50 1.4 nM), followed by gamma-MSH (IC50 7 nM). alpha-MSH, beta-MSH and ACTH-(1-39) were significantly less potent, with IC50 values of 30, 19 and 21 nM respectively. ACTH-(4-10) was poorly active (IC50 2.4 microM), while corticotropin-like intermediate lobe peptide (CLIP) and beta-endorphin were totally ineffective. The recombinant receptor was found to stimulate adenylate cyclase. The potency order of the agonists in this assay was consistent with that of the binding displacement assays. This receptor represents the orthologue of the human melanocortin 3 receptor reported recently. The growing family of melanocortin receptors constitute the molecular basis for the variety of actions of melanocortins that have been described over the years. The availability of functionally expressed receptors from the melanocortin family will allow the development of a specific pharmacology, and a better understanding of the function of the pro-opiomelanocortin-derived peptides. Images Figure 6 PMID

  2. Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues.

    PubMed

    Gao, Zhifang; Maurousset, Laurence; Lemoine, Remi; Yoo, Sang-Dong; van Nocker, Steven; Loescher, Wayne

    2003-04-01

    The acyclic polyol sorbitol is a primary photosynthetic product and the principal photosynthetic transport substance in many economically important members of the family Rosaceace (e.g. almond [Prunus dulcis (P. Mill.) D.A. Webber], apple [Malus pumila P. Mill.], cherry [Prunus spp.], peach [Prunus persica L. Batsch], and pear [Pyrus communis]). To understand key steps in long-distance transport and particularly partitioning and accumulation of sorbitol in sink tissues, we have cloned two sorbitol transporter genes (PcSOT1 and PcSOT2) from sour cherry (Prunus cerasus) fruit tissues that accumulate large quantities of sorbitol. Sorbitol uptake activities and other characteristics were measured by heterologous expression of PcSOT1 and PcSOT2 in yeast (Saccharomyces cerevisiae). Both genes encode proton-dependent, sorbitol-specific transporters with similar affinities (K(m) sorbitol of 0.81 mM for PcSOT1 and 0.64 mM for PcSOT2). Analyses of gene expression of these transporters, however, suggest different roles during leaf and fruit development. PcSOT1 is expressed throughout fruit development, but especially when growth and sorbitol accumulation rates are highest. In leaves, PcSOT1 expression is highest in young, expanding tissues, but substantially less in mature leaves. In contrast, PcSOT2 is mainly expressed only early in fruit development and not in leaves. Compositional analyses suggest that transport mediated by PcSOT1 and PcSOT2 plays a major role in sorbitol and dry matter accumulation in sour cherry fruits. Presence of these transporters and the high fruit sorbitol concentrations suggest that there is an apoplastic step during phloem unloading and accumulation in these sink tissues. Expression of PcSOT1 in young leaves before completion of the transition from sink to source is further evidence for a role in determining sink activity. PMID:12692316

  3. Molecular cloning and characterization of a group II chaperonin delta-subunit from soybean.

    PubMed

    Nong, Van Hai; Arahira, Masaomi; Phan, Van Chi; Kim, Chan-Shick; Zhang, Deyu; Udaka, Kyoko; Fukazawa, Chikafusa

    2002-08-01

    Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta). This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.

  4. Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element

    PubMed Central

    Goldberg, M.L.; Paro, R.; Gehring, W.J.

    1982-01-01

    We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling ˜60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the “white proximal region”, thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype. ImagesFig. 2.Fig. 4.Fig. 5.Fig. 6. PMID:16453411

  5. Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism.

    PubMed

    Wüthrich, K L; Bovet, L; Hunziker, P E; Donnison, I S; Hörtensteiner, S

    2000-01-01

    Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.

  6. Human GluR6 kainate receptor (GRIK2): Molecular cloning, expression, polymorphism, and chromosomal assignment

    SciTech Connect

    Paschen, W.; Blackstone, C.D.; Huganir, R.L. ); Ross, C.A. Max-Planck-Institute for Neurological Research, Koeln )

    1994-04-01

    Glutamate receptors mediate the majority of excitatory neurotransmission in the brain, and molecular cloning studies have revealed several distinct families. Because neuropathological states and possibly human disorders may involve kainate-preferring glutamate receptors, the authors have isolated a cDNA clone for the human GluR6 kainate-preferring receptor. This clone shows a very high sequence similarity with that of the rat, except for a part of the 3[prime] untranslated region in which there is a TAA triplet repeat. When the protein was overexpressed in human embryonic kidney 293 cells, it had a molecular weight, an antibody recognition, and a glutamate ligand-binding profile similar to those of the rate GluR6 receptor. Northern analysis showed expression in both human cerebral and cerebellar cortices. By PCR analysis of rodent-human monochromosomal cell lines, the human GluR6 could be assigned to chromosome 6. The length of the TAA triplet repeat was polymorphic in the normal population, with at least four alleles and an observed heterozygosity of about 45%. These studies should provide the basis for expression or linkage studies of the GluR6 kainate receptor in human disease or neuropathologic states. 53 refs., 7 figs.

  7. A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

    PubMed

    Wei, Xiaomu; Das, Jishnu; Fragoza, Robert; Liang, Jin; Bastos de Oliveira, Francisco M; Lee, Hao Ran; Wang, Xiujuan; Mort, Matthew; Stenson, Peter D; Cooper, David N; Lipkin, Steven M; Smolka, Marcus B; Yu, Haiyuan

    2014-12-01

    Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

  8. A dedicated database program for cataloging recombinant clones and other laboratory products of molecular biology technology.

    PubMed

    Jenson, H B

    1989-06-01

    A novel computer database program dedicated to storing, cataloging, and accessing information about recombinant clones and libraries has been developed for the IBM (or compatible) personal computer. This program, named CLONES, also stores information about bacterial strains and plasmid and bacteriophage vectors used in molecular biology. The advantages of this method are improved organization of data, fast and easy assimilation of new data, automatic association of new data with existing data, and rapid retrieval of desired records using search criteria specified by the user. Individual records are indexed in the database using B-trees, which automatically index new entries and expedite later access. The use of multiple windows, pull-down menus, scrolling pick-lists, and field-input techniques make the program intuitive to understand and easy to use. Daughter databases can be created to include all records of a particular type, or only those records matching user-specified search criteria. Separate databases can also be merged into a larger database. This computer program provides an easy-to-use and accurate means to organize, maintain, access, and share information about recombinant clones and other laboratory products of molecular biology technology. PMID:2631777

  9. Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid cotransporter.

    PubMed Central

    Hagenbuch, B; Meier, P J

    1994-01-01

    We have used a cDNA probe from a cloned rat liver Na+/taurocholate cotransporting polypeptide (Ntcp) to screen a human liver cDNA library. A 1,599-bp cDNA clone that encodes a human Na+/taurocholate cotransporting polypeptide (NTCP) was isolated. The human NTCP consists of 349 amino acids (calculated molecular mass of 38 kD) and exhibits 77% amino acid homology with the rat Ntcp. In vitro translation experiments indicate that the protein is glycosylated and has a molecular weight similar to the rat Ntcp. Injection of in vitro transcribed cRNA into Xenopus laevis oocytes resulted in the expression of Na(+)-dependent taurocholate uptake. Saturation kinetics indicated that the human NTCP has a higher affinity for taurocholate (apparent Km = 6 microM) than the previously cloned rat protein (apparent Km = 25 microM). NTCP-mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives (100 microM), bumetanide (500 microM), and bromosulphophthalein (100 microM). Southern blot analysis of genomic DNA from a panel of human/hamster somatic cell hybrids mapped the human NTCP gene to chromosome 14. PMID:8132774

  10. [Molecular cloning and expression analysis of an Aux/IAA gene (RgIAA1) from Rehmannia glutinosa].

    PubMed

    Wang, Feng-Qing; Tian, Yun-He; Li, Ming-Jie; Yang, Jin-Feng; Zhang, Bao; Lin, Wen-Xiong; Chen, Xin-Jian; Zhang, Zhong-Yi

    2013-12-01

    To clone and analyze a member of the Auxin/indole-3-acetic acid (Aux/IAA) gene family, RgIAA1, from Rehmannia glutinosa. The transcriptional EST database of R. glutinosa was used to clone the new Aux/IAA gene by cDNA probe of AtIAA14. Bioinformatics was applied to analyze the sequence characteristics of RgIAA1 protein and construct phylogenetiC trees. Quantitative RT-PCR has been applied to detect the transcription level of RgIAA1 in seven tissues as well as in leaves under three stresses. The results showed that, the cDNA sequence of RgIAA1 contains 903 bp was obtained. The open reading frame (ORF) of RgIAA1 was 681 bp encoding 226 amino acids, which has typical structural domains and characteristic sequence of Aux/IAA family proteins. RgIAA1 showed the highest expression level in unfolded leaf, followed by the stem. And the expression of RglAA1 was quickly decreased with leaf growing up. The transcription level increased under continuous cropping conditions while it reduced both in salinity and waterlogging stresses. RgIAA1, an Aux/IAA gene from R. glutinosa has been obtained for the first time, which can lay the foundation for further studies about its molecular function in development and responses to stress.

  11. Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (Penaeus monodon).

    PubMed

    Jiang, Shigui; Qiu, Lihua; Zhou, Falin; Huang, Jianhua; Guo, Yihui; Yang, Keng

    2009-01-01

    The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5' untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3' UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37 degrees C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.

  12. Molecular cloning and mRNA expression analysis of interleukin-8 gene in Japanese sea perch (Lateolabrax japonicus).

    PubMed

    Qiu, Lihua; Zhang, Hanhua; Yang, Keng; Jiang, Shigui

    2009-05-01

    Interleukin-8 (IL-8), the first known chemokine, is a CXC chemokine, which is cable of attracting neutrophils and inducing them to release lysozomal enzymes, triggering the respiratory burst. In the present study, the cDNA of an IL-8 was cloned from Japanese sea perch Lateolabrax japonicus (designated LjIL-8) by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of LjIL-8 consisted of 803 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 300 bp encoding a polypeptide of 99 amino acid residues with a predicted molecular weight of 6.6 kDa. The high identity of LjIL-8 with IL-8 in other organisms indicated that LjIL-8 should be a new member of the IL-8 family. By fluorescent quantitative real-time PCR, mRNA transcript of LjIL-8 was detectable in all the examined tissues with higher level in spleen and head-kidney. The temporal expression of LjIL-8 mRNA in the spleen was up-regulated by lipopolyssacharide (LPS) stimulation and reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. These results indicated that LjIL-8 was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of L. japonicus.

  13. Molecular cloning and characterization of a cyclin B gene on the ovarian maturation stage of black tiger shrimp (Penaeus monodon).

    PubMed

    Qiu, Lihua; Jiang, Shigui; Zhou, Falin; Huang, Jianhua; Guo, Yihui

    2007-01-24

    The techniques of homology cloning and anchored PCR were used to clone the cyclin B gene from black tiger shrimp. The full length cDNA of black tiger shrimp cyclin B (btscyclin B) contained a 5' untranslated region (UTR) of 102 bp, an ORF of 1,206 bp encoding a polypeptide of 401 amino acids with an estimated molecular mass of 45 kDa and a 3' UTR of 396 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btscyclin B was homological to the cyclin B of other species and even the mammalians. Two conserved signature sequences of cyclin B gene family were found in the btscyclin B deduced amino acid sequence. The temporal expressions of cyclin B gene in the different tissues, including liver, ovary, muscle, brain stomach, heart and intestine, were measured by RT-PCR. mRNA expression of cyclin B could be detected in liver, ovary, muscle, brain, stomach, heart and strongest in the ovary, but almost not be detected in the intestine. In ovarian maturation stages, the expression of btscyclin B was different. The result indicated that btscyclin B was constitutive expressed and played an important role in the cell division stage.

  14. Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

    1994-01-01

    Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

  15. Molecular Cloning and Gene Expression of Canine Apoptosis Inhibitor of Macrophage

    PubMed Central

    TOMURA, Shintaro; UCHIDA, Mona; YONEZAWA, Tomohiro; KOBAYASHI, Masato; BONKOBARA, Makoto; ARAI, Satoko; MIYAZAKI, Toru; TAMAHARA, Satoshi; MATSUKI, Naoaki

    2014-01-01

    Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM. PMID:25649949

  16. Molecular cloning of Mu d(bla lacZ) transcriptional and translational fusions.

    PubMed Central

    Wanner, B L

    1987-01-01

    The vector pBW2 was made to selectively clone chimeric plasmids with chromosomal Mu d(bla lacZ) transcriptional or translational fusions. It was tetracycline resistant and had the carboxyl-terminal end of bla distal to its PstI site. Because ligation of PstI-digested chromosomal DNA of a Mu d(bla lacZ) insertion with pBW2 restored bla, ampicillin-resistant chimeric plasmids were selectable. These plasmids had the Mu d bla amino terminus and simultaneously acquired other Mu d sequences including lacZ, the chromosomal fusion joint, and the DNA adjacent to the nearest chromosomal PstI site. The plasmid pBW2 was useful in the molecular cloning of several psi and pho::lacZ(Mu d) fusions, as well as chromosomal genes located near Mu d insertions. PMID:3032905

  17. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs.

    PubMed

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs.

  18. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs.

    PubMed

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs. PMID:27010315

  19. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs

    PubMed Central

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs. PMID:27010315

  20. Extensive TCR junctional diversity of V gamma 9/V delta 2 clones from human female reproductive tissues.

    PubMed

    Christmas, S E; Brew, R; Thornton, S M; Deniz, G; Flanagan, B F

    1995-09-01

    Panels of gamma delta T cell clones bearing the V gamma 9/V delta 2 form of TCR were derived from human first trimester decidualized endometrium and cervix. Seventy-three percent of these clones expressed the human mucosal lymphocyte Ag HML-1 compared with only 14% of PBL V gamma 9/V delta 2 clones, indicating that most clones were derived from the tissue itself rather than contaminating peripheral blood. All 13 clones isolated expressed V gamma 9JPC gamma 1- and V delta 2(D)J delta 1-encoded receptors; TCR gamma and delta junctional regions from most of these were sequenced and analyzed, together with the TCR-delta junctional region of a sequence obtained from bulk CD3+ decidual leukocytes. There was considerable junctional diversity of both gamma- and delta-chains with a similar extent of germline V and J gene trimming and N-region nucleotide addition to that found in PBL V gamma 9/V delta 2 cells. Eight of eleven TCR-delta junctional sequences contained a strongly hydrophobic amino acid in position 97, as has been found in > 90% o V gamma 9/V delta 2 clones. Thymic V gamma 9/V delta 2 cells show much less junctional diversity and less pronounced selection at residue 97 of the delta-chain. Thus, unlike the mouse, gamma delta T cells from human female reproductive tissues exhibit extensive TCR junctional as well as combinatorial diversity. This suggests that V gamma 9/V delta 2 cells in these human tissues have undergone selective but diverse peripheral expansion in response to antigenic stimuli in a similar manner to those in peripheral blood.

  1. Mitochondrial pyruvate dehydrogenase. Molecular cloning of the E1 alpha subunit and expression analysis.

    PubMed Central

    Grof, C P; Winning, B M; Scaysbrook, T P; Hill, S A; Leaver, C J

    1995-01-01

    A polymerase chain reaction-based approach was used to isolate cDNA clones encoding the E1 alpha subunit of the mitochondrial pyruvate dehydrogenase from higher plants. Putative full-length clones were identified on the basis of similarity to E1 alpha sequences from nonplant sources. Southern blot analysis revealed a small family of genes in potato (Solanum tuberosum L.), whereas in cucumber (Cucumis sativus) there are only one or two genes. Tissue-specific variation in the relative amounts of E1 alpha mRNA was observed in northern blot analysis of different potato tissues, with the highest steady-state transcript levels found in floral tissue. Measurement of pyruvate dehydrogenase activity in cucumber cotyledons showed that there is a transient increase to a maximum at 4 to 5 d postimbibition. Western blot analysis revealed that the amount of E1 alpha protein also peaks at this time. Steady-state transcript levels in germinating cucumber cotyledons also show transient accumulation, peaking 2 d postimbibition. These data are consistent with regulation of E1 alpha at the level of transcription and/or mRNA stability in postgerminative cucumber cotyledons. PMID:7659754

  2. Embryonic rather than extraembryonic tissues have more impact on the development of placental hyperplasia in cloned mice.

    PubMed

    Miki, H; Wakisaka, N; Inoue, K; Ogonuki, N; Mori, M; Kim, J-M; Ohta, A; Ogura, A

    2009-06-01

    Somatic cell cloning by nuclear transfer (NT) in mice is associated with hyperplastic placentas at term. To dissect the effects of embryonic and extraembryonic tissues on this clone-associated phenotype, we constructed diploid (2n) fused with (<-->) tetraploid (4n) chimeras from NT- and fertilization-derived (FD) embryos. Generally, the 4n cells contributed efficiently to all the extraembryonic tissues but not to the embryo itself. Embryos constructed by 2n NT<-->4n FD aggregation developed hyperplastic placentas (0.33+/-0.22 g) with a predominant contribution by NT-derived cells. Even when the population of FD-derived cells in placentas was increased using multiple FD embryos (up to four) for aggregation, most placentas remained hyperplastic (0.36+/-0.13 g). By contrast, placentas of the reciprocal combination, 2n FD<-->4n NT, were less hyperplastic (0.15+/-0.02 g). These nearly normal-looking placentas had a large proportion of NT-derived cells. Thus, embryonic rather than extraembryonic tissues had more impact on the onset of placental hyperplasia, and that the abnormal placentation in clones occurs in a noncell-autonomous manner. These findings suggest that for improvement of cloning efficiency we should understand the mechanisms regulating placentation, especially those of embryonic origin that might control the proliferation of trophoblastic lineage cells.

  3. Molecular cloning and characterization of an achene-seed-specific promoter from motherwort (Leonurus japonicus Houtt).

    PubMed

    Xie, Chengjian; Zhang, Beibei; Wang, De; Kou, Fei; Zhao, Xupeng; Yang, Xingyong

    2011-01-01

    LJAMP1 is a small antimicrobial protein purified previously from the seeds of motherwort, and it is expressed preferentially in seeds. A 794-bp upstream sequence of the ATG start codon was isolated using a genome walking method and cloned into the upstream of the β-glucuronidase (GUS) reporter gene to determine the GUS tissue-specific expression pattern. The transgenic tobacco showed that pLJAMP1 promoter derived GUS reporter gene special expression in pollen, achene and seed. The analysis of cis-acting elements also revealed pLJAMP1 promoter contained pollen and seed related transcriptional control elements.

  4. Purification, characterization and molecular cloning of human hepatic lysosomal acid lipase.

    PubMed

    Ameis, D; Merkel, M; Eckerskorn, C; Greten, H

    1994-02-01

    Lysosomal acid lipase (LAL) is a hydrolase essential for the intracellular degradation of cholesteryl esters and triacylglycerols. This report describes a multi-step procedure for the purification of LAL from human liver. After solubilization with non-ionic detergent, acid hydrolase activity was purified 17000-fold to apparent homogeneity by sequential chromatography on Concanavalin A Sepharose, carboxymethyl-cellulose, phenyl Superose, Mono S cation exchange and Superose 12 gel-filtration columns. This procedure yielded two silver-staining protein bands of 56 kDa and 41 kDa on SDS/PAGE. Size-exclusion chromatography of the 41-kDa protein indicated that the enzyme was catalytically competent as a monomer of approximately 38 kDa. When assayed in the presence of cholesteryl oleate or trioleoylglycerol, purified acid lipase had Vmax values of 4390 nmol fatty acid.min-1.mg protein and 4756 nmol fatty acid.min-1.mg protein-1, and apparent Km values of 0.142 mM and 0.138 mM, respectively. The purified enzyme was most active at low pH (4.5-5.0) and required non-ionic detergent and ethylene glycol for optimal stability. Incubation of the 41-kDa acid lipase with endoglucosaminidase H reduced the molecular mass by 4-6 kDa, demonstrating Asn-linked glycosylation with high-mannose oligosaccharides. Deglycosylation did not affect enzymic activity, indicating that carbohydrates are not required for LAL activity. Based on partial peptide sequence, an oligonucleotide was synthesized and utilized to isolate LAL cDNA clones from a human liver cDNA library. A full-length LAL cDNA contained 2626 nucleotides and coded for a predicted protein of 372 amino acids, preceded by a 27 residue hydrophobic signal peptide. Hepatic LAL differed from fibroblast acid lipase at the N-terminus and revealed extensive similarities with human gastric lipase and rat lingual lipase, confirming a gene family of acid lipases. Northern hybridization using the complete LAL cDNA as a radiolabeled probe

  5. Molecular cloning, characterization and expression of goose Toll-like receptor 5.

    PubMed

    Fang, Qiang; Pan, Zhiming; Geng, Shizhong; Kang, Xilong; Huang, Jinlin; Sun, Xiaolin; Li, Qiuchun; Cai, Yinqiang; Jiao, Xinan

    2012-10-01

    Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are vital to activation of the innate immune system in response to invading pathogens through their recognition of pathogen-associated molecular patterns (PAMPs). TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the goose TLR5 gene using rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of goose TLR5 cDNA is 2583 bp in length and encodes an 860 amino acid protein. The entire coding region of the TLR5 gene was successfully amplified from genomic DNA and contained a single exon. The putative amino acid sequence of goose TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR-CT) domain, a transmembrane domain and an intracellular Toll-interleukin-1 receptor (TIR) domain. The amino acid sequence of goose TLR5 shared 50.5% identity with human (Homo sapiens), 49.8% with mouse (Mus musculus) and 82.7% with chicken (Gallus gallus). The goose TLR5 gene was highly expressed in the spleen, liver and brain; moderately expressed in PBMCs, kidney, lung, heart, bone marrow, small intestine and large intestine; and minimally expressed in the cecum. HEK293 cells transfected with goose TLR5 and NF-κB-luciferase containing plasmids significantly responded to flagellin from Salmonella typhimurium indicating that it is a functional TLR5 homologue. In response to infection with S. enterica serovar Enteritidis (SE), the level of TLR5 mRNA significantly increased over the control in PBMCs at 1 d post infection (p.i.) and was slightly elevated in the spleen at 1 d or 3 d p.i. IL-6 was expressed below control levels in PBMCs but was upregulated in the spleen. In contrast to IL-6, an evident decrease in the expression level of IL-8 was observed in both PBMCs and spleens at 1 d or 3 d p.i. SE challenge also resulted in an increase in the mRNA expression of IL-18 and IFN-γ in PBMCs

  6. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    USGS Publications Warehouse

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  7. Molecular cloning, expression analysis, and functional characterization of connexin44.1: a zebrafish lens gap junction protein.

    PubMed

    Cason, N; White, T W; Cheng, S; Goodenough, D A; Valdimarsson, G

    2001-06-01

    The connexin family of genes codes for proteins that oligomerize into a connexon of six subunits to form one half of the gap junction channel. Gap junctions are plasma membrane structures that mediate intercellular communication by joining the cytoplasm of two cells, allowing the passage of small molecules and metabolites, and contributing significantly to the maintenance of tissue homeostasis. The signaling mediated by these junctions appears to be necessary for the correct timing of key developmental events. This communication is especially important in the avascular lens where the intercellular passage of metabolites, second messengers, and ions is necessary to maintain the correct ionic balance in the lens fibre cells, and prevent cataract formation. To characterize the role that the connexin genes play in development, a novel connexin was cloned from zebrafish. A genomic clone was isolated that contained a 1,173 base open reading frame. The nucleotide sequence in this open reading frame shows extensive sequence similarity to mouse connexin50 (Cx50), chicken Cx45.6, sheep Cx49, and human Cx50. The protein encoded by this open reading frame contains 391 amino acids, with a predicted molecular weight of 44.1 kDa and a typical connexin transmembrane topology. By using the LN54 radiation hybrid panel, the Cx44.1 gene was mapped to linkage group 1. Whole-mount in situ hybridization and Northern blot analyses were performed on zebrafish embryos at various developmental stages to characterize the developmental expression of the Cx44.1 message. The ocular lens was the only tissue in which Cx44.1 transcripts were detected. The transcripts were first detected in the lens around 24 hr post fertilization and remained detectable until 120 hr post fertilization. Electrophysiological analysis of Cx44.1 channels revealed gating properties that were virtually identical to the mouse and chicken orthologues of Cx44.1.

  8. Molecular cloning and sequencing of the cDNA of cop1 gene from Pisum sativum.

    PubMed

    Zhao, L; Wang, C; Zhu, Y; Zhao, J; Wu, X

    1998-02-11

    Cop1 protein plays an important role in seedling development of higher plants. The cDNA of cop1 gene from pea (Pisum sativum) was cloned and sequenced. Cop1 protein of pea is predicted to have 672 amino acids and a molecular mass of 76 kDa. Sequence comparison between Cop1 proteins of pea and Arabidopsis thaliana revealed that the two Cop1 proteins were highly homologous in the regions with functional domains and at the C-terminus. Immunoblotting performed with polyclonal antibodies against recombinant Cop1 of pea showed that Cop1 protein was present in seedlings germinated both in light and darkness.

  9. Molecular cloning of the mitogenic mannose/maltose-specific rhizome lectin from Calystegia sepium.

    PubMed

    Van Damme, E J; Barre, A; Verhaert, P; Rougé, P; Peumans, W J

    1996-11-18

    cDNA clones encoding the mitogenic mannose/maltose-specific lectin from the rhizomes of hedge bindweed (Calystegia sepium) have been isolated and sequenced. Comparison of the deduced amino acid sequence and the molecular weight of the lectin subunit as determined by mass spectrometry indicated that the mature protein comprises the entire open reading frame of the cDNA, which implies that the primary translation product contains no signal peptide and is not proteolytically processed. Searches in the databases revealed sequence homology with the previously described lectins from the taxonomically unrelated Moraceae species Artocarpus integrifolia and Maclura pomifera.

  10. Molecular cloning and functional analysis of a 10-epi-junenol synthase from Inula hupehensis.

    PubMed

    Gou, Jun-Bo; Li, Zhen-Qiu; Li, Chang-Fu; Chen, Fang-Fang; Lv, Shi-You; Zhang, Yan-Sheng

    2016-09-01

    Junenol based-eudesmanolides have been detected in many compositae plant species and were reported to exhibit various pharmacological activities. So far, the gene encoding junenol synthase has never been isolated. Here we report the molecular cloning and functional analysis of a 10-epi-junenol synthase from Inula hupehensis (designated IhsTPS1). IhsTPS1 converts the substrate farnesyl diphosphate into multiple sesquiterpenes with the product 10-epi-junenol being predominant. The transcript levels of IhsTPS1 correlate well with the accumulation pattern of 10-epi-junenol in I. hupehensis organs, supporting its biochemical roles in vivo. PMID:27231873

  11. Molecular cloning, expression, and sequence of the pilin gene from nontypeable Haemophilus influenzae M37.

    PubMed Central

    Coleman, T; Grass, S; Munson, R

    1991-01-01

    Nontypeable Haemophilus influenzae M37 adheres to human buccal epithelial cells and exhibits mannose-resistant hemagglutination of human erythrocytes. An isogenic variant of this strain which was deficient in hemagglutination was isolated. A protein with an apparent molecular weight of 22,000 was present in the sodium dodecyl sulfate-polyacrylamide gel profile of sarcosyl-insoluble proteins from the hemagglutination-proficient strain but was absent from the profile of the isogenic hemagglutination-deficient variant. A monoclonal antibody which reacts with the hemagglutination-proficient isolate but not with the hemagglutination-deficient isolate has been characterized. This monoclonal antibody was employed in an affinity column for purification of the protein as well as to screen a genomic library for recombinant clones expressing the gene. Several clones which contained overlapping genomic fragments were identified by reaction with the monoclonal antibody. The gene for the 22-kDa protein was subcloned and sequenced. The gene for the type b pilin from H. influenzae type b strain MinnA was also cloned and sequenced. The DNA sequence of the strain MinnA gene was identical to that reported previously for two other type b strains. The DNA sequence of the strain M37 gene is 77% identical to that of the type b pilin gene, and the derived amino acid sequence is 68% identical to that of the type b pilin. Images PMID:1673447

  12. [Molecular targeted drugs for soft tissue sarcoma and neuroendocrine tumor].

    PubMed

    Kato, Shunsuke

    2015-08-01

    Both the soft tissue sarcomas and the neuroendocrine tumors are rare diseases. Therefore the recruiting of these patients was more difficult than other cancer species, and the development of the new therapy for these diseases did not readily advance. However, the identification of driver molecules for each sub-type enabled us to the development of the molecular targeted drugs. As for the GIST, several TKIs are used, but in late years it is found that susceptibility of TKIs varies according to difference in second mutation. In this chapter, the molecular target drug for the soft tissue sarcoma and the neuroendocrine tumor is reviewed. PMID:26281696

  13. Molecular cloning and expression analysis of a pearl oyster (Pinctada martensii) heat shock protein 90 (HSP90).

    PubMed

    Liang, H Y; Wang, Z X; Lei, Q N; Huang, R L; Deng, Y W; Wang, Q H; Jiao, Y; Du, X D

    2015-01-01

    Heat shock protein 90 (HSP90) is an important molecular chaperone required for proper folding of cellular proteins, and thus, it plays an essential role in protecting cells from damage during stress. In this study, an HSP90 cDNA designated PmHSP90 was cloned from the mantle tissue of the pearl oyster Pinctada martensii using reverse transcription polymerase chain reaction (RT-PCR) coupled with the rapid amplification of cDNA ends (RACE) approach. PmHSP90 cDNA was 2584 bp in length, including an open reading frame of 2160 bp, which encodes a polypeptide of 719 amino acid residues, with predicted molecular mass and isoelectric point of 83.0 kDa and 4.87, respectively. Multiple-sequence alignment indicated that HSP90 is highly conserved among species, and PmHSP90 showed 89% sequence identity to Crassostrea gigas HSP90. Five conserved amino acid blocks defined as HSP90 protein family signatures were also observed in PmHSP90, indicating that PmHSP90 may be a cytosolic member of the HSP90 family. Expression levels of PmHSP90 were detected in various tissues of P. martensii and in hemocytes under three different stress conditions using quantitative real-time PCR (qPCR). The results demonstrate that PmHSP90 mRNA is constitutively expressed in all the tested tissues and may be involved in the immune response against thermal stress, lipopolysaccharide stimulation, and nucleus insertion operations. Studies on PmHSP90 are a valuable source to further explore the immune system in pearl oysters during the production of pearls, and may enhance our knowledge of molluscan innate immunity. PMID:26782528

  14. Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase.

    PubMed Central

    Saito, K; Miura, N; Yamazaki, M; Hirano, H; Murakoshi, I

    1992-01-01

    Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach. Images PMID:1518833

  15. Molecular cloning and functional expression of a brain-specific somatostatin receptor.

    PubMed Central

    Bruno, J F; Xu, Y; Song, J; Berelowitz, M

    1992-01-01

    The PCR and conventional library screening were used to clone the brain-specific somatostatin receptor rSSTR-4 from a rat genomic library. The deduced amino acid sequence encodes a protein of 384 amino acids and displays structural and sequence homologies with members of the G protein-receptor superfamily. The amino acid sequence of rSSTR-4 is 60% and 48% identical to that of somatostatin receptors SSTR-1 and SSTR-2, respectively, two recently cloned subtypes. Competition curve analysis of the binding properties of the receptor transiently expressed in COS-1 cells revealed a higher apparent affinity for somatostatin 14 than for somatostatin 28. In contrast, the somatostatin analogs SMS 201-995, IM 4-28, and MK-678 failed to displace specific binding in transfected cells. These characteristics resemble the pharmacological binding properties of the previously described brain-specific somatostatin-receptor subtype. Examination of the tissue distribution of mRNA for rSSTR-4 revealed expression limited to various brain regions with highest levels in the cortex and hippocampus. Thus, based on the pharmacology and tissue localization of this receptor, we conclude that rSSTR-4 represents a brain-specific somatostatin receptor. Images PMID:1360663

  16. Molecular cloning and expression of two HSP70 genes in the prawn, Macrobrachium rosenbergii

    PubMed Central

    Liu, Jun; Yang, Wei-Jun; Zhu, Xiao-Jing; Karouna-Renier, Natalie K.; Rao, Ranga K.

    2004-01-01

    Two complementary deoxyribonucleic acid (cDNA) clones encoding 2 different 70-kDa heat shock proteins (HSPs) were isolated from the prawn Macrobrachium rosenbergii. The cDNA clones were 2448 and 2173 bp in length and contained 1950- and 1734-bp open reading frames (ORFs), respectively. The ORFs encoded 649– and 577–amino acid polypeptides, which were named Mar-HSC70 and Mar-HSP70, respectively, according to the sequence identities with other known HSC70s and HSP70s and based on their inducibility in response to heat shock stress (at 35°C). Genomic DNA sequence analysis revealed no introns in either gene. The major structural differences between the 2 proteins were a 60–amino acid segment and a 14–amino acid segment present in the N-terminal and C-terminal, respectively, of Mar-HSC70 that were not found in Mar-HSP70. Northern blotting and semiquantitative reverse transcription–polymerase chain reaction analyses indicated that the Mar-HSP70 gene was expressed under heat shock (35°C) stress in a non–tissue-specific manner. In contrast, Mar-HSC70 messenger ribonucleic acid was constitutively expressed in every tissue except muscle, and its expression in response to heat shock (at 35°C) changed only in muscle. PMID:15544169

  17. Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

    PubMed Central

    Henriques, Ana; Rodríguez-Caballero, Arancha; Criado, Ignacio; Langerak, Anton W.; Nieto, Wendy G.; Lécrevisse, Quentin; González, Marcos; Cortesão, Emília; Paiva, Artur; Almeida, Julia; Orfao, Alberto

    2014-01-01

    Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes. PMID:24488564

  18. Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

    NASA Astrophysics Data System (ADS)

    Li, Jiakai; Wu, Xiangwei; Tan, Jing; Zhao, Ruixiang; Deng, Lingwei; Liu, Xiande

    2015-07-01

    P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 cDNA designated PtHSP20 was cloned from P. textile. The full-length cDNA of PtHSP20 is 1 090 bp long and contains a 5' untranslated region (UTR) of 93 bp, a 3' UTR of 475 bp, and an open reading frame (ORF) of 522 bp. The PtHSP20 cDNA encodes 173 amino acid residues and has a molecular mass of 20.22 kDa and an isoelectric point of 6.2. Its predicted amino acid sequence shows that PtHSP20 contains a typical α-crystallin domain (residues 77-171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, PtHSP20 shows moderate homology to other mollusk sHSPs. PtHSP20 mRNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of PtHSP20 mRNA was down-regulated in all of the tissues except the adductor muscle and gonad.

  19. From Lesions to Viral Clones: Biological and Molecular Diversity amongst Autochthonous Brazilian Vaccinia Virus

    PubMed Central

    Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Neto, José Diomedes; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

    2015-01-01

    Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment. PMID:25785515

  20. Molecular cloning and nucleotide sequence of a transforming gene detected by transfection of chicken B-cell lymphoma DNA

    NASA Astrophysics Data System (ADS)

    Goubin, Gerard; Goldman, Debra S.; Luce, Judith; Neiman, Paul E.; Cooper, Geoffrey M.

    1983-03-01

    A transforming gene detected by transfection of chicken B-cell lymphoma DNA has been isolated by molecular cloning. It is homologous to a conserved family of sequences present in normal chicken and human DNAs but is not related to transforming genes of acutely transforming retroviruses. The nucleotide sequence of the cloned transforming gene suggests that it encodes a protein that is partially homologous to the amino terminus of transferrin and related proteins although only about one tenth the size of transferrin.

  1. Molecular cloning and characterization of five opsin genes from the marine flatfish Atlantic halibut (Hippoglossus hippoglossus).

    PubMed

    Helvik, J V; Drivenes, O; Naess, T H; Fjose, A; Seo, H C

    2001-01-01

    Most molecular studies on the visual system in fish have been performed on freshwater teleosts such as goldfish and zebrafish where cones and rods appear simultaneously. Many marine fishes have long larval phase in the upper pelagic zone before transformation into a juvenile and a benthic life style. The retina at the larval stages consists of only single cone cells; later during metamorphosis double cones and rods develop. The flatfish Atlantic halibut (Hippoglossus hippoglossus) is a typical example of a marine species with such a two-step retina development. In this study, we have cloned five different opsins from Atlantic halibut larvae and juvenile retinas. Sequence comparisons with other opsins and phylogenetic analysis show that the five genes belong to the opsins of long-wavelength sensitive (L); middle-wavelength sensitive, M(Cone) and M(Rod); and short-wavelength sensitive, S(Blue) and S(Ultraviolet), respectively. In situ hybridization analysis reveals expression in double cone (L and M(Cone)), single cone (S(Blue) and S(Ultraviolet)), and rod (M(Rod)) types of photoreceptor cells in juvenile halibut retina. The visual system in Atlantic halibut seems therefore to have all four types of cone photoreceptors in addition to rod photoreceptors. This work shows for the first time molecular isolation of a complete set of retinal visual pigment genes from a marine teleost and describes the first cloning of an ultraviolet-sensitive opsin type from a marine teleost.

  2. Molecular and physiological mechanisms regulating tissue reunion in incised plant tissues.

    PubMed

    Asahina, Masashi; Satoh, Shinobu

    2015-05-01

    Interactions among the functionally specialized organs of higher plants ensure that the plant body develops and functions properly in response to changing environmental conditions. When an incision or grafting procedure interrupts the original organ or tissue connection, cell division is induced and tissue reunion occurs to restore physiological connections. Such activities have long been observed in grafting techniques, which are advantageous not only for agriculture and horticulture but also for basic research. To understand how this healing process is controlled and how this process is initiated and regulated at the molecular level, physiological and molecular analyses of tissue reunion have been performed using incised hypocotyls of cucumber (Cucumis sativus) and tomato (Solanum lycopersicum) and incised flowering stems of Arabidopsis thaliana. Our results suggest that leaf gibberellin and microelements from the roots are required for tissue reunion in the cortex of the cucumber and tomato incised hypocotyls. In addition, the wound-inducible hormones ethylene and jasmonic acid contribute to the regulation of the tissue reunion process in the upper and lower parts, respectively, of incised Arabidopsis stems. Ethylene and jasmonic acid modulate the expression of ANAC071 and RAP2.6L, respectively, and auxin signaling via ARF6/8 is essential for the expression of these transcription factors. In this report, we discuss recent findings regarding molecular and physiological mechanisms of the graft union and the tissue reunion process in wounded tissues of plants.

  3. Cloning, tissue and ontogenetic expression of the taurine transporter in the flatfish Senegalese sole (Solea senegalensis).

    PubMed

    Pinto, Wilson; Rønnestad, Ivar; Jordal, Ann-Elise Olderbakk; Gomes, Ana S; Dinis, Maria Teresa; Aragão, Cláudia

    2012-04-01

    Flatfish species seem to require dietary taurine for normal growth and development. Although dietary taurine supplementation has been recommended for flatfish, little is known about the mechanisms of taurine absorption in the digestive tract of flatfish throughout ontogeny. This study described the cloning and ontogenetic expression of the taurine transporter (TauT) in the flatfish Senegalese sole (Solea senegalensis). Results showed a high similarity between TauT in Senegalese sole and other vertebrates, but a change in TauT amino acid sequences indicates that taurine transport may differ between mammals and fish, reptiles or birds. Moreover, results showed that Senegalese sole metamorphosis is an important developmental trigger to promote taurine transport in larvae, especially in muscle tissues, which may be important for larval growth. Results also indicated that the capacity to uptake dietary taurine in the digestive tract is already established in larvae at the onset of metamorphosis. In Senegalese sole juveniles, TauT expression was highest in brain, heart and eye. These are organs where taurine is usually found in high concentrations and is believed to play important biological roles. In the digestive tract of juveniles, TauT was more expressed in stomach and hindgut, indicating that dietary taurine is quickly absorbed when digestion begins and taurine endogenously used for bile salt conjugation may be recycled at the posterior end of the digestive tract. Therefore, these results suggest an enterohepatic recycling pathway for taurine in Senegalese sole, a process that may be important for maintenance of the taurine body levels in flatfish species.

  4. Goldfish neurokinin B: Cloning, tissue distribution, and potential role in regulating reproduction.

    PubMed

    Qi, Xin; Zhou, Wenyi; Li, Shuisheng; Liu, Yun; Ye, Gang; Liu, Xiaochun; Peng, Chun; Zhang, Yong; Lin, Haoran

    2015-09-15

    Neurokinin B (NKB) is a member of the tackykinin (TAC) family known to play a critical role in the neuroendocrine regulation of reproduction in mammals. However, its biological functions in teleosts are less clear. The aim of this study was to determine the role of NKB in fish reproduction using goldfish as a model. Two transcripts, TAC3a and TAC3b, which encode several NKBs, including NKBa-13, NKBa-10, NKBb-13, and NKBb-11, were cloned. Phylogenetic analysis revealed that NKBa-10 and NKBb-11 are closely related to mammalian NKB, while NKB-13s are more conserved in teleosts. Quantitative real-time PCR analyses in various tissues showed that TAC3a and TAC3b mRNAs were mainly expressed in the brain. In situ hybridization further detected TAC3a and TAC3b mRNAs in several regions of the brain known to be involved in the regulation of reproduction and metabolism, as well as in the neurohypophysis of the pituitary. To investigate the potential role of NKBs in reproduction, goldfish were injected intraperitoneally with synthetic NKBa-13, -10, NKBb-13, or -11 peptides and the mRNA levels of hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary gonadotropin subunits were measured. NKBa-13, -10, or NKBb-13, but not -11, significantly increased hypothalamic salmon GnRH and pituitary FSHβ and LHβ mRNA levels in both female and male goldfish. Finally, ovariectomy increased, while estradiol replacement reduced, TAC3a mRNA levels without affecting TAC3b expression in the hypothalamus. These data suggest that NKBa-13, -10, and NKBb-13 play a role in mediating the estrogen negative feedback regulation of gonadotropins.

  5. Identification of porcine polycystic kidney disease 1 (PKD1) gene: molecular cloning, expression profile, and implication in disease model.

    PubMed

    He, Jin; Wang, Qingsong; Ye, Jianhua; Hu, Xiaoxiang; Li, Ning

    2011-12-15

    The polycystic kidney disease 1 (PKD1) gene, which accounts for ~85% of human autosomal dominant polycystic kidney disease (ADPKD) cases, has been extensively studied in human and mouse. Much information about the pathogenesis of and treatments for ADPKD has been gained from the use of mouse models. However, because mouse models pose some limitations, further studies in other model systems are needed to investigate the biological basis of ADPKD. The pig is regarded as an important biomedical model. Thus, we isolated a pig PKD1 homolog and characterized its cDNA sequence, genomic structure, expression profile, alternative splicing, methylation status, protein characteristics, and immunohistochemical features in both neonatal and adult pigs. The pig PKD1 cDNA is 14,209bp long and encodes a 4305-residue polypeptide. The genomic sequence of PKD1 is ~50kb with 46 exons. An alternative splice acceptor site was identified in intron 9. PKD1 is expressed in all tissues tested in both neonatal and adult pigs and exhibits a developmentally regulated expression pattern. Western blotting revealed that the molecular mass of polycystin-1 is ~460kDa, but its expression level is relatively low. Immunohistochemical study of the kidneys shows that polycystin-1 is mainly expressed in the tubular epithelia. Bisulfite methylation analysis of CpG islands in the promoter region does not show a direct correlation between methylation status and expression level among different tissues/cells. The cloning and characterization of pig PKD1 indicates that the pig and human genes are highly similar in length of genomic and cDNA sequences, genomic structure and context, expression patterns, conserved transcription factor binding sites, and the molecular mass of the encoded polycystin-1. These data support our current understanding of PKD1, and suggest that the pig is an ideal candidate for development of an ADPKD disease model. PMID:21945688

  6. Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N

    SciTech Connect

    McAllister, G.; Amara, S.G.; Lerner, M.R. )

    1988-07-01

    Sera from some patients with systemic lupus erythematosus and other autoimmune diseases have antibodies against nuclear antigens. An example is anti-Sm sera, which recognize proteins associated with small nuclear RNA molecules (small nuclear ribonucleoprotein (snRNP) particles). In this paper anti-Sm sera were used to probe immunoblots of various rat tissues. A previously unidentified M{sub r} 28,000 polypeptide was recognized by these anti-Sm sera. This polypeptide, referred to as N, is expressed in a tissue-specific manner, being most abundant in rat brain, less so in heart, and undetectable in the other tissues examined. Immunoprecipitation experiments using antibodies directed against the cap structure of small nuclear RNAs have demonstrated that N is a snRNP-associated polypeptide. Anti-Sm serum was also used to isolate a partial cDNA clone ({lambda}rb91) from a rat brain phage {lambda}gt11 cDNA expression library. A longer cDNA clone was obtained by rescreening the library with {lambda}rb91. In vitro transcription and subsequent translation of this subcloned, longer insert (pGMA2) resulted in a protein product with the same electrophoretic and immunological properties as N, confirming that pGMA2 encodes N. The tissue distribution of N and the involvement of snRNP particles in nuclear pre-mRNA processing may imply a role for N in tissue-specific pre-mRNA splicing.

  7. Molecular cloning, mRNA expression and enzymatic characterization of cathepsin F from olive flounder (Paralichthys olivaceus).

    PubMed

    Ahn, Sang Jung; Kim, Na Young; Seo, Jung Soo; Je, Ju Eun; Sung, Ji Hea; Lee, Sang Hwan; Kim, Moo-Sang; Kim, Joong Kyun; Chung, Joon Ki; Lee, Hyung Ho

    2009-10-01

    Cathepsin F is a recently described papain-like cysteine protease of unknown function, and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. In the present study, the cDNA of olive flounder (Paralichthys olivaceus) cathepsin F (PoCtF) was cloned by the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) approaches. The PoCtF gene was determined to consist of the 1844 bp nucleotide sequence which encodes for a 475-amino acid polypeptide. The results of RT-PCR analysis revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however the PoCtF expressions increased significantly in gill at 3h post-injection with lipopolysaccharide (LPS). Also, immunostaining using anti-PoCtF antibody was strongest on the epidermal mucus in the fin. The cDNA encoding mature enzyme of PoCtF was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC, a substrate commonly used for functional characterization of cysteine proteinases, and the optimal pH for the protease activity was 7.5. The findings of the present study suggest that PoCtF has a higher optimum pH than mammalian cathepsin F, and PoCtF is an interesting target for future investigations of the role of cathepsin F in the epidermal mucus and fish innate immune system. PMID:19545641

  8. Molecular cloning and mRNA expression of cathepsin C gene in black tiger shrimp (Penaeus monodon).

    PubMed

    Qiu, Lihua; Jiang, Shigui; Huang, Jianhua; Wang, Weifang; Zhang, Dianchang; Wu, Qiaer; Yang, Keng

    2008-07-01

    Cathepsin C (dipeptidyl-peptidase I, DPPI) is a lysosomal cysteine proteinase belonging to the papain superfamily, which is capable of removing dipeptides sequentially from the amino terminus of peptide and protein substrates. In the present study, the cDNA of a cathepsin C was cloned from black tiger shrimp Penaeus monodon (designated PmcathepsinC) by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of PmcathepsinC consisted of 2051 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 1350 bp encoding a polypeptide of 449 amino acid residues with a predicted molecular weight of 50.0 kDa and theoretical isoelectric point of 5.65. The high identity of PmcathepsinC with Cathepsin C in other organisms indicated that PmcathepsinC should be a new member of the Cathepsin C family. By fluorescent quantitative real-time PCR, mRNA transcript of PmcathepsinC was detectable in all the examined tissues with higher level in ovary and heart. The temporal expression of PmcathepsinC mRNA in the hepatopancreas was up-regulated by lipopolysaccharide (LPS) stimulation and reached the maximum level at 4 h post-stimulation, and then dropped back to the original level gradually. These results indicated that PmcathepsinC was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of P. monodon.

  9. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1.

    PubMed Central

    Collman, R; Balliet, J W; Gregory, S A; Friedman, H; Kolson, D L; Nathanson, N; Srinivasan, A

    1992-01-01

    We isolated and molecularly cloned a human immunodeficiency virus type 1 (HIV-1) strain (89.6) which is unusual because it is both macrophage-tropic and extremely cytopathic in lymphocytes. Moreover, this is the first well-characterized infectious molecularly cloned macrophage-tropic HIV-1 strain derived from peripheral blood. HIV-1 89.6 differs markedly from other macrophage-tropic isolates within the envelope V3 region, which is important in determining cell tropism and cytopathicity. HIV-1 89.6 may thus represent a transitional isolate between noncytopathic macrophage-tropic viruses and cytopathic lymphocyte-tropic viruses. Images PMID:1433527

  10. Molecular cloning and expression analysis of five GhRAXs in upland cotton (Gossypium hirsutum L.).

    PubMed

    Dai, T C; Wang, Z M

    2015-10-05

    The formation of axillary meristems in leaf axils is a prerequisite for the development of lateral shoots, which largely contribute to plant architecture. Several transcription factor-encoding genes, including CUC3, RAX, LAS, LOF1, and ROX, have been cloned by screening for axillary meristem mutants in Arabidopsis thaliana. These genes will facilitate our understanding of the mechanisms underlying axillary meristem development. In this study, we report the cloning of five genes from cotton (Gossypium hirsutum L.) that are orthologous to A. thaliana REGULATORS OFAXILLARY MERISTEMS (RAX) and tomato Blind (Bl), and they are designated GhRAX1, 2, 3, 4, and 5. Sequence analyses indicated that all five genes shared conserved protein domains with RAX and Bl. Phylogenetic analyses of protein sequences revealed that GhRAX2/3/4 were close to RAX1, whereas GhRAX1 and GhRAX5 were close to RAX3. Expression patterns of these genes in different tissues were also analyzed using real-time PCR.

  11. Thermal and molecular investigation of laser tissue welding

    SciTech Connect

    Small, W., IV

    1998-06-01

    Despite the growing number of successful animal and human trials, the exact mechanisms of laser tissue welding remain unknown. Furthermore, the effects of laser heating on tissue on the molecular scale are not fully understood. To address these issues, a multi-front attack oil both extrinsic (solder/patch mediated) and intrinsic (laser only) tissue welding was launched using two-color infrared thermometry, computer modeling, weld strength assessment, biochemical assays, and vibrational spectroscopy. The coupling of experimentally measured surface temperatures with the predictive numerical simulations provided insight into the sub-surface dynamics of the laser tissue welding process. Quantification of the acute strength of the welds following the welding procedure enabled comparison among trials during an experiment, with previous experiments, and with other studies in the literature. The acute weld integrity also provided an indication of tile probability of long-term success. Molecular effects induced In the tissue by laser irradiation were investigated by measuring tile concentrations of specific collagen covalent crosslinks and characterizing the Fourier-Transform infrared (FTIR) spectra before and after the laser exposure.

  12. Molecular Structure of Frizzled, a Drosophila Tissue Polarity Gene

    PubMed Central

    Adler, P. N.; Vinson, C.; Park, W. J.; Conover, S.; Klein, L.

    1990-01-01

    The function of the frizzled (fz) locus is required to coordinate the cytoskeletons of pupal epidermal cells so that a parallel array of cuticular hairs and bristles is produced. We report here the molecular cloning and characterization of the fz locus. The locus is very large. Mutations that inactivate the gene are spread over 100 kb of genomic DNA. The major mRNA product of the gene is a 4-kb RNA that is encoded by 5 exons spread over more than 90 kb of genomic DNA. Conceptual translation of this mRNA indicates that it encodes an integral membrane protein that is likely to contain both extracellular and cytoplasmic domains. PMID:2174014

  13. Molecular cloning of rat homologues of the Drosophila melanogaster dunce cAMP phosphodiesterase: evidence for a family of genes.

    PubMed Central

    Swinnen, J V; Joseph, D R; Conti, M

    1989-01-01

    To study the structure and function of cyclic nucleotide phosphodiesterases (PDEs) involved in mammalian gametogenesis, a rat testis cDNA library was screened at low stringency with a cDNA clone coding for the Drosophila melanogaster dunce-encoded PDE as a probe. This screening resulted in the isolation of two groups of cDNA clones, differing in their nucleotide sequences (ratPDE1 and ratPDE2). In the rat testis, RNA transcripts corresponding to both groups of clones were expressed predominantly in germ cells. Additional screenings of a Sertoli cell cDNA library with a ratPDE2 clone as a probe led to the isolation of two more groups of clones (rat-PDE3 and ratPDE4). Unlike ratPDE1 and ratPDE2, these clones hybridized to transcripts present predominantly in the Sertoli cell. In the middle of the coding region, all four groups of clones were homologous to each other. The deduced amino acid sequences of part of this region were also homologous to the D. melanogaster dunce PDE and to PDEs from bovine and yeast. These data indicate that a family of genes homologous to the D. melanogaster dunce-encoded PDE is present in the rat and that these genes are differentially expressed in somatic and germ cells of the seminiferous tubule. These findings provide a molecular basis for the observed heterogeneity of cAMP PDEs. Images PMID:2546153

  14. Multiscale mechanobiology: mechanics at the molecular, cellular, and tissue levels.

    PubMed

    Guo, Chin-Lin; Harris, Nolan C; Wijeratne, Sithara S; Frey, Eric W; Kiang, Ching-Hwa

    2013-06-03

    Mechanical force is present in all aspects of living systems. It affects the conformation of molecules, the shape of cells, and the morphology of tissues. All of these are crucial in architecture-dependent biological functions. Nanoscience of advanced materials has provided knowledge and techniques that can be used to understand how mechanical force is involved in biological systems, as well as to open new avenues to tailor-made bio-mimetic materials with desirable properties.In this article, we describe models and show examples of how force is involved in molecular functioning, cell shape patterning, and tissue morphology.

  15. Molecular cloning, expression analysis, and function of decorin in goat ovarian granulosa cells.

    PubMed

    Peng, J Y; Gao, K X; Xin, H Y; Han, P; Zhu, G Q; Cao, B Y

    2016-10-01

    Decorin (DCN), a component of the extracellular matrix (ECM), participates in ECM assembly and influences cell proliferation and apoptosis in many mammalian tissues and cells. However, expression and function of DCN in the ovary remain unclear. This study cloned the full-length cDNA of goat DCN obtained from the ovary of an adult goat. Sequence analysis revealed that the putative DCN protein shared a highly conserved amino acid sequence with known mammalian homologs. The tissue distribution of DCN mRNA expression was evaluated by real-time PCR, and the results showed that DCN was widely expressed in the tissues of adult goat. Immunohistochemistry results suggested that DCN protein existed in the granulosa cells and oocytes from all types of follicles and theca cells of antral follicles. Moreover, hCG-induced DCN mRNA expression was significantly reduced by the inhibitors of protein kinase A, PI3K, or p38 kinase (P < 0.05), which are key mediators involved in hCG-induced DCN expression. Overexpression of DCN significantly increased apoptosis and blocked cell cycle progression in cultured granulosa cells (P < 0.05). Western blot analysis also showed that overexpression of DCN upregulated the expression levels of p21 protein (P < 0.05), whereas no effects were observed on the expression of Bax and Bcl-2 and on Bcl-2/Bax ratio (P > 0.05). These findings suggested that DCN regulates the apoptosis and cell cycle of granulosa cells. PMID:27565237

  16. Molecular cloning and primary structure of human glial fibrillary acidic protein

    SciTech Connect

    Reeves, S.A.; Helman, L.J.; Allison, A.; Israel, M.A. )

    1989-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate-filament (IF) protein that is highly specific for cells of astroglial lineage, although its tissue-specific role is speculative. Determination of the primary structure of this protein should be of importance for understanding the functional role it plays in astroglia. Therefore, the authors isolated a cDNA clone encoding this protein and determined its nucleotide sequence. The predicted amino acid sequence indicates that GFAP shares structural similarities-particularly in the central rod domain and to a lesser degree in the carboxyl-terminal domain-with other IF proteins found in nonepithelial cell types. Considerable sequence divergence in the amino-terminal region of GFAP suggests that the tissue-specific functions of this IF protein might be mediated through this region of the molecule. In contrast, conservation of structural characteristics and a moderate degree of sequence conservation in the carboxyl-terminal region suggest functional similarities. Blot hybridization analysis using the GFAP cDNA as a probe failed to detect GFAP mRNA in both normal and neoplastic human tissues in which IF proteins other than GFAP are known to be expressed.

  17. Development of a mutant strain of Escherichia coli for molecular cloning of highly methylated DNA

    SciTech Connect

    Bishr, M.A.

    1991-01-01

    A mutant strain of Escherichia coli designated as GR219 that allows efficient molecular cloning of highly methylated bean DNA has been developed by UV light mutation of the parent LE392 str[sup r] strain. This mutant strain, like the parent, is streptomycin resistant and is biologically contained, because it requires thymidine for growth. Both the wild type and the mutant strain have lambda phage receptors so both can be utilized for construction of genomic libraries using the phase as a vector. The efficiency of transformation of the parent and the mutant strain with a recombinant plasmid containing bean DNA was compared to the efficiency of transformation of the PLK-F[prime] strain, which has a deletion of mcrA and mcrB genes and, therefore, allows transformation with methylated bean DNA. It has been found that the GR219 strain has the highest efficiency of transformation, while the PLK-F[prime] strain shows less, and the parent LE392 str[sup r] strain the least efficiency of transformation. These results indicate that strains of E. coli with mcrA and mcrB genes can recognize and degrade highly methylated DNA. However, other undefined factors affected by the altered gene(s) in the GR219 strain are also involved in the recognition and degradation of any cloned foreign DNA.

  18. Cloning and molecular modelling of pectin degrading glycosyl hydrolase of family 28 from soil metagenomic library.

    PubMed

    Sathya, T A; Jacob, Ani Methew; Khan, Mahejibin

    2014-01-01

    Western Ghats of India is recognized as one of the 12 mega diversity regions of the world and is the hot spot for unrevealed microbial diversity. To explore the diversity of polysaccharide degrading enzymes in that region, metagenomic library was constructed from forest soil of Southern Western Ghats region. Nine pectinolytic clones with the ability to degrade citrus pectin were isolated based on function based screening of the library. Sequence analysis of pg_4 clone containing revealed that it contained GH family 28 domain (pfam00295) belonging to polygalacturonase superfamily (PLN03003). Its amino acid sequence analysis showed 25-55 % identity to the other well-characterized polygalacturonases. Molecular modeling of pg_4 revealed that it comprised of three right handed-parallel β sheets, one anti-parallel β sheet and one α helix with three conserved catalytic residue D 2263, D 284-85 and H 312 at the C terminal end. The enzyme characterized was able to hydrolyze both apple and citrus pectin with K m values of 1.685 and 1.542 mg ml(-1) and retained more that 80 % of activity at pH 5-9 and temperature 20-60 °C.

  19. Molecular cloning, structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum.

    PubMed

    Wischang, Diana; Radlow, Madlen; Schulz, Heiko; Vilter, Hans; Viehweger, Lutz; Altmeyer, Matthias O; Kegler, Carsten; Herrmann, Jennifer; Müller, Rolf; Gaillard, Fanny; Delage, Ludovic; Leblanc, Catherine; Hartung, Jens

    2012-10-01

    The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (k(cat) = 153 s(-1), 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata. PMID:22884431

  20. Molecular cloning and expression of the Leishmania tropica KMP-11 gene.

    PubMed

    Meriee, Mouayad; Soukkarieh, Chadi; Abbady, Abdul Qader A

    2014-08-01

    Kinetoplastid membrane protein-11 (KMP-11) is a small protein of 11 kDa present in all kinetoplastid protozoa studded so far. This protein which is highly expressed in all stages of the Leishmania life cycle is considered a potential candidate for a leishmaniasis vaccine against many leishmania species. KMP-11 has been recently described in Leishmania tropica. In the present study, the KMP-11 gene was extracted from L. tropica by PCR using two oligonucleotide primers designed to amplify the entire coding region of this gene. Then, the purified PCR products were successfully ligated into a high expression vector the pRSET-GFP. This expression vector provides the opportunity to clone the desired insert as a fusion protein with a GFP and a tag, polyhistidine region. The GFP use as a carrier to improve immune response and the polyhistidine tag facilitates detection of the expressed protein with anti-His antibodies and also purification of the protein using affinity purification. After wards KMP-11 coding region was sequenced and the recombinant protein was induced and purified from Escherichia coli cultures. The results of the present study will increase our knowledge about molecular cloning and expression of the L. tropica KMP-11 gene, and this may be used as an effective target for controlling cutenous leishmaniasis. PMID:25597146

  1. Molecular cloning, expression, and regulation of the ovalbumin gene in pigeon oviduct epithelial cells.

    PubMed

    Zhang, H; Lu, L Z; Chen, L; Tao, Z R; Chen, F; Zhong, S L; Liu, Y L; Tian, Y; Yan, P S

    2014-01-10

    The full-length pigeon ovalbumin (OVA) gene cDNA was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends. A 386-amino acid protein was predicted for the obtained sequence, which had 67% identity with the chicken protein. Similar to chicken OVA, the pigeon OVA gene is a non-inhibitory serine protease inhibitor. Quantitative PCR analysis revealed that pigeon OVA mRNA was highly expressed in the oviduct, and trace amounts were detected in other tissues. During the reproductive cycle, pigeon oviduct OVA mRNA expression reached its peak during the egg-laying stage, decreased with brooding, and then increased again during the squab-feeding period. Moreover, the relative OVA expression level in pigeon oviduct epithelial cells could be upregulated by a constant concentration of steroid hormones.

  2. Molecular Cloning and Characterisation of Farnesyl Pyrophosphate Synthase from Tripterygium wilfordii

    PubMed Central

    Zhao, Yu-Jun; Chen, Xin; Zhang, Meng; Su, Ping; Liu, Yu-Jia; Tong, Yu-Ru; Wang, Xiu-Juan; Huang, Lu-Qi; Gao, Wei

    2015-01-01

    Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products. PMID:25938487

  3. Molecular cloning of α-2-macroglobulin from hemocytes of common periwinkle Littorina littorea.

    PubMed

    Borisova, Elena A; Gorbushin, Alexander M

    2014-08-01

    We report the sequence of the proteinase inhibitor with a wide inhibition spectrum, α-2-macroglobulin (α2M), belonging to the thioester superfamily of proteins. This is the first α2M sequence from coenogastropod prosobranch snails. The full-length cDNA was cloned by RACE method, spans 7897 bp and contains an open reading frame of 5460 bp. The ORF encodes a protein of 1819 amino acids. The deduced mature protein contains 1795 amino acids with a molecular weight of 200 kDa and isoelectric point of 5.00. Littorina littorea α2M bears 4 conserved α2M domains and one internal thioester. Phylogenetic analysis showed that the sequence forms well supported cluster with Mollusca species and other representatives of Lophotrochozoa. PMID:24830774

  4. Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase.

    PubMed Central

    James, E R; McLean, D C; Perler, F

    1994-01-01

    Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD sequence and is 3 nt longer. All residues involved in metal ion binding, active site formation, folding, and dimer formation in SODs are conserved. Data indicate the OvEcSOD and O. volvulus cytosolic SOD are separate gene products and that the OvEcSOD appears to possess the characteristics of a membrane-bound or secreted enzyme which may be involved in the parasite defense against phagocyte-generated reactive oxygen species. Images PMID:8300230

  5. Production of Bovine Embryos and Calves Cloned by Nuclear Transfer Using Mesenchymal Stem Cells from Amniotic Fluid and Adipose Tissue.

    PubMed

    da Silva, Carolina Gonzales; Martins, Carlos Frederico; Cardoso, Tereza Cristina; da Cunha, Elisa Ribeiro; Bessler, Heidi Christina; Martins, George Henrique Lima; Pivato, Ivo; Báo, Sônia Nair

    2016-04-01

    The less differentiated the donor cells are used in nuclear transfer (NT), the more easily are they reprogrammed by the recipient cytoplasm. In this context, mesenchymal stem cells (MSCs) appear as an alternative to donor nuclei for NT. The amniotic fluid and adipose tissue are sources of MSCs that have not been tested for the production of cloned embryos in cattle. The objective of this study was to isolate, characterize, and use MSCs derived from amniotic fluid (MSC-AF) and adipose tissue (MSC-AT) to produce cloned calves. Isolation of MSC-AF was performed using in vivo ultrasound-guided transvaginal amniocentesis, and MSC-AT were isolated by explant culture. Cellular phenotypic and genotypic characterization by flow cytometry, immunohistochemistry, and RT-PCR were performed, as well as induction in different cell lineages. The NT was performed using MSC-AF and MSC-AT as nuclear donors. The mesenchymal markers of MSC were expressed in bovine MSC-AF and MSC-AT cultures, as evidenced by flow cytometry, immunohistochemistry, and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes, and adipocytes. Embryo production was similar between the cell types, and two calves were born. The calf from MSC-AT was born healthy, and this fact opens a new possibility of using this type of cell to produce cloned cattle by NT. PMID:27055630

  6. Molecular cloning and expression of the human delta7-sterol reductase.

    PubMed

    Moebius, F F; Fitzky, B U; Lee, J N; Paik, Y K; Glossmann, H

    1998-02-17

    Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Delta7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith-Lemli-Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Delta7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7-8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 microM), BM15766 (IC50 1.2 microM), and triparanol (IC50 14 microM). Our work paves the way to clarify whether a defect in the delta7-sterol reductase gene underlies the Smith-Lemli-Opitz syndrome. PMID:9465114

  7. Molecular cloning and expression of the human Δ7-sterol reductase

    PubMed Central

    Moebius, Fabian F.; Fitzky, Barbara U.; Lee, Joon No; Paik, Young-Ki; Glossmann, Hartmut

    1998-01-01

    Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Δ7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith–Lemli–Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Δ7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7–8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 μM), BM15766 (IC50 1.2 μM), and triparanol (IC50 14 μM). Our work paves the way to clarify whether a defect in the Δ7-sterol reductase gene underlies the Smith–Lemli–Opitz syndrome. PMID:9465114

  8. Molecular cloning of the alpha-globin transcription factor CP2.

    PubMed Central

    Lim, L C; Swendeman, S L; Sheffery, M

    1992-01-01

    CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1. Images PMID:1732747

  9. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

    PubMed

    Jiménez-Vázquez, Eric N; Díaz-Velásquez, Clara E; Uribe, R M; Arias, Juan M; García, Ubaldo

    2016-02-01

    Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.

  10. Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency

    SciTech Connect

    Suzuki, Y.; Aoki, Y.; Ishida, Y.

    1994-09-01

    Holocarboxylase synthetase (HCS) catalyzes biotin incorporation into various carboxylases that require biotin as a prosthetic group. They are acetyl-CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis; pyruvate carboxylase, a key enzyme of gluconeogenesis; propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase, enzymes involved in amino acid catabolism. HCS is therefore involved in various metabolic processes and is a key enzyme for biotin utilization by mammalian cells. Deficiency of HCS in man is known to cause biotin-responsive multiple carboxylase deficiency. Isolation of cDNA clones for the enzyme is essential to understand HCS and its deficiency at the molecular level. We purified bovine liver HCS and sequenced its proteolytic peptides. Degenerative oligonucleotide primers were synthesized from the two peptide sequences and used to amplify a putative HCS cDNA fragment from human liver by PCR. Using the amplified DNA fragment as a probe, we screened {lambda}gt10 human liver cDNA library and isolated 12 positive clones. The isolated cDNAs encoded a protein of 726 amino acids with molecular mass of 80,759. The protein contained several sequences identical or similar to those of peptides derived from the bovine liver HCS. The predicted protein had a homologous region with BirA which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. We expressed the protein using pET3 a vector in E. coli (BL21 strain) and raised antiserum against the expressed protein. The antiserum immunoprecipitated HCS activities of human lymphoblasts and bovine liver. A one-base deletion and a missense mutation were found in cells from siblings with HCS deficiency. The human HCS gene was assigned to chromosome 21, region 21q22.1 by fluorescence in situ hybridization analysis.

  11. Characterization of nonprimate hepacivirus and construction of a functional molecular clone

    PubMed Central

    Scheel, Troels K. H.; Kapoor, Amit; Nishiuchi, Eiko; Brock, Kenny V.; Yu, Yingpu; Andrus, Linda; Gu, Meigang; Renshaw, Randall W.; Dubovi, Edward J.; McDonough, Sean P.; Van de Walle, Gerlinde R.; Lipkin, W. Ian; Divers, Thomas J.; Tennant, Bud C.; Rice, Charles M.

    2015-01-01

    Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3′-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3′-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3′-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel hepacivirus in its natural host. PMID:25646476

  12. Effective serological and molecular screening of deceased tissue donors.

    PubMed

    Kitchen, A D; Newham, J A; Gillan, H L

    2013-12-01

    A comprehensive and effective screening programme is essential to support the banking of tissues from deceased donors. However, the overall quality of the samples obtained from deceased donors, quantity and condition, is often not ideal, and this may lead to problems in achieving accurate and reliable results. Additionally a significant percentage of referrals are still rejected upon receipt as unsuitable for screening. We are actively involved in improving the overall quality of deceased donor screening outcomes, and have specifically evaluated and validated both serological and molecular assays for this purpose, as well as developing a specific screening strategy to minimise the specificity issues associated with serological screening. Here we review the nature and effectiveness of the deceased donor screening programme implemented by National Health Service Blood and Transplant (NHSBT), the organisation with overall responsibility for the supply of tissue products within England. Deceased donor screening data, serological and molecular, from August 2007 until May 2012 have been collated and analysed. Of 10,225 samples referred for serology screening, 5.5 % were reported as reactive; of 2,862 samples referred for molecular screening, 0.1 % were reported as reactive/inhibitory. Overall 20 % of the serological and 100 % of the molecular screen reactivity was confirmed as reflecting true infection. The use of a sequential serology screening algorithm has resulted in a marked reduction of tissues lost unnecessarily due to non-specific screen reactivity. The approach taken by NHSBT has resulted in the development of an effective and specific approach to the screening of deceased tissue donors. PMID:23354598

  13. The 5′ Untranslated Region of a Novel Infectious Molecular Clone of the Dicistrovirus Cricket Paralysis Virus Modulates Infection

    PubMed Central

    Kerr, Craig H.; Wang, Qing S.; Keatings, Kathleen; Khong, Anthony; Allan, Douglas; Yip, Calvin K.

    2015-01-01

    ABSTRACT Dicistroviridae are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species, Cricket paralysis virus (CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro-transcribed RNA from the CrPV clone into Drosophila Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adult Drosophila flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5′ untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells. IMPORTANCE Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host

  14. Molecular cloning and expression analysis of GABA(A) receptor-associated protein (GABARAP) from small abalone, Haliotis diversicolor.

    PubMed

    Bai, Rongyao; You, Weiwei; Chen, Jun; Huang, Heqing; Ke, Caihuan

    2012-10-01

    GABA(A) receptor-associated protein (GABARAP), a multifunctional protein participating in autophagy process, is evolutionarily conserved and involves in innate immunity in eukaryotic cells, but currently there is no research on the relationship between GABARAP and innate immunity in mollusc. In the present study, the GABARAP full-length cDNA and its genomic DNA were firstly cloned from small abalone (Haliotis diversicolor), which was named as saGABARAP. Its full-length cDNA is 963 bp with a 354 bp open reading frame encoding a protein of 117 aa, a 276 bp 5'-UTR, and a 333 bp 3'-UTR including a poly(A) tail, two typical polyadenylation signals (AATAA) and two RNA instability motifs (ATTTA). The deduced protein has an estimated molecular weight of 13.9 kDa and a predicted PI of 8.73. Its genomic DNA comprises 4352 bp, containing three exons and two introns. Quantitative real-time PCR analysis revealed that saGABARAP was constitutively expressed in all examined tissues, with the highest expression level in hepatopancreas, and was upregulated in hepatopancreas and hemocytes after bacterial challenge. In addition, saGABARAP was ubiquitously expressed at all examined embryonic and larval development stages. These results suggested that saGABARAP could respond to bacteria challenge and may play a vital role in the adult innate immune system against pathogens and the development process of abalone embryo and larvae.

  15. Molecular Cloning, Expression Analysis, and Preliminarily Functional Characterization of the Gene Encoding Protein Disulfide Isomerase from Jatropha curcas.

    PubMed

    Wang, Haibo; Zou, Zhurong; Gong, Ming

    2015-05-01

    Reactive oxygen species (ROS) in plants, arising from various environmental stresses, impair the thiol-contained proteins that are susceptible to irregular oxidative formation of disulfide bonds, which might be alleviated by a relatively specific modifier called protein disulfide isomerase (PDI). From our previous data of the transcriptome and digital gene expression of cold-hardened Jatropha curcas, a PDI gene was proposed to be cold-relevant. In this study, its full-length cDNA (JcPDI) was cloned, with the size of 1649 bp containing the entire open reading frame (ORF) of 1515 bp. This ORF encodes a polypeptide of 504 amino acids with theoretical molecular weight of 56.6 kDa and pI value of 4.85. One N-terminal signal peptide (-MASKGSIWSCMFLFSLI VAISAGEG-) and the C-terminal anchoring sequence motif (-KDEL-) specific to the endoplasmic reticulum, as well as two thioredoxin domains (-CGHC-), are also found by predictions. Through semi-quantitative RT-PCR, the expression of JcPDI was characterized to be tissue-differential strongly in leaves and roots, but weakly in stems, and of cold-induced alternations. Furthermore, JcPDI overexpression in yeast could notably enhance the cold resistance of host cells. Conclusively, these results explicitly suggested a considerable association of JcPDI to cold response and a putative application potential for its correlated genetic engineering. PMID:25825250

  16. Molecular cloning and functional characterization of a high-affinity zinc importer (DrZIP1) from zebrafish (Danio rerio).

    PubMed

    Qiu, Andong; Shayeghi, Majid; Hogstrand, Christer

    2005-06-15

    Zinc is a vital micronutrient to all organisms and a potential toxicant to aquatic animals. It is therefore of importance to understand the mechanism of zinc regulation. In the present study, we molecularly cloned and functionally characterized a zinc transporter of the SLC39A family [commonly referred to as the ZIP (Zrt- and Irt-related protein) family] from the gill of zebrafish (Danio rerio) (DrZIP1). DrZIP1 protein was found to localize at the plasma membrane and to function as a zinc uptake transporter when being expressed in either chinook salmon (Oncorhynchus tshawytscha) embryonic 214 cells or Xenopus laevis oocytes. In comparison with pufferfish transporter proteins (FrZIP2 and FrECaC) that are known to facilitate cellular zinc uptake, DrZIP1 appears to have high affinity to bind and transport zinc, suggesting that it maybe a high-affinity zinc uptake transporter (Km < 0.5 microM) in fish. Orthologues of DrZIP1 were also identified in both freshwater and seawater pufferfish (Tetraodon nigroviridis and Takifugu rubripes), indicating that these proteins may be functionally conserved among different fish species. DrZIP1 mRNA is expressed in all the tissues examined in the present study and thus DrZIP1 may be a constitutive zinc uptake transporter in many cell types of zebrafish.

  17. Molecular cloning and characterization of four caspases members in Apostichopus japonicus.

    PubMed

    Shao, Yina; Li, Chenghua; Zhang, Weiwei; Duan, Xuemei; Li, Ye; Jin, Chunhua; Xiong, Jinbo; Qiu, Qiongfen

    2016-08-01

    The caspase family representing aspartate-specific cysteine proteases have been demonstrated to possess key roles in apoptosis and immune response. We previously demonstrated that LPS challenged Apostichopus japonicus coelomocyte could significantly induced apoptosis in vitro. However, apoptosis related molecules were scarcely investigated in this economic species. In the present work, we cloned and characterized four members caspase family from A. japonicus (designated as Ajcaspase-2, Ajcaspase-3, Ajcaspase-6, and Ajcaspase-8, respectively) by RACE. Multiple sequence alignment and structural analysis revealed that all Ajcaspases contained the conservative CASC domain at C terminal, in which some unique features for each Ajcaspase made them different from each other. These specific domains together with phylogenetic analysis supported that all these four identified proteins belonged to novel members of apoptotic signaling pathway in sea cucumber. Tissue distribution analysis revealed that four Ajcaspase genes were constitutively expressed in all examined tissues. The expression of Ajcaspase-2 was tightly correlated with that of Ajcaspase-8 in each detected tissues. Ajcaspase-3 and Ajcaspase-6 transcripts were both highly expressed in immune tissue of coelomocytes. Furthermore, the Vibrio splendidus challenged sea cucumber coelomocytes could significantly up-regulate the mRNA expressions of four genes. The expression levels of Ajcaspase-2 and Ajcaspase-8 were relative earlier than those of Ajcaspase-6 and Ajcaspase-3, respectively, which could be inferred that Ajcapase-2 might directly modulate Ajcaspase-6, and Ajcaspase-8 initiate the expression of Ajcaspase-3. The induce expressions differed among each Ajcaspase depending upon their roles such as initiator or effector caspase. All our results demonstrated that four Ajcaspases present diversified functions in apoptotic cascade signaling pathway of sea cucumber under immune response. PMID:27245866

  18. Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis.

    PubMed

    Asawatreratanakul, Kasem; Zhang, Yuan-Wei; Wititsuwannakul, Dhirayos; Wititsuwannakul, Rapepun; Takahashi, Seiji; Rattanapittayaporn, Atiya; Koyama, Tanetoshi

    2003-12-01

    Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration. The enzyme responsible for the cis-1,4-polymerization of isoprene units has been idengified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis. Sequence analysis revealed that all of the five highly conserved regions among cis-prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis-prenyltransferase. Northern blot analysis indicated that the transcript(s) of the Hevea cis-prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined. In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 x 103-1 x 104 Da. Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased. These results suggest that the Hevea cis-prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber.

  19. Datura stramonium agglutinin: cloning, molecular characterization and recombinant production in Arabidopsis thaliana.

    PubMed

    Nishimoto, Keisuke; Tanaka, Kaori; Murakami, Takahiro; Nakashita, Hideo; Sakamoto, Hikaru; Oguri, Suguru

    2015-02-01

    Datura stramonium seeds contain at least three chitin-binding isolectins [termed Datura stramonium agglutinin (DSA)] as homo- or heterodimers of A and B subunits. We isolated a cDNA encoding isolectin B (DSA-B) from an immature fruit cDNA library; this contained an open reading frame encoding 279 deduced amino acids, which was confirmed by partial sequencing of the native DSA-B peptide. The sequence consisted of: (i) a cysteine (Cys)-rich carbohydrate-binding domain composed of four conserved chitin-binding domains and (ii) an extensin-like domain of 37 residues containing four SerPro4-6 motifs that was inserted between the second and third chitin-binding domains (CBDs). Although each chitin-binding domain contained eight conserved Cys residues, only the second chitin-binding domain contained an extra Cys residue, which may participate in dimerization through inter-disulfide bridge formation. Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, the molecular mass of homodimeric lectin composed of two B-subunits was determined as 68,821 Da. The molecular mass of the S-pyridilethylated B-subunit were found to be 37,748 Da and that of the de-glycosylated form was 26,491 Da, which correlated with the molecular weight estimated from the deduced sequence. Transgenic Arabidopsis plants overexpressing the dsa-b demonstrated hemagglutinating activity. Recombinant DSA-B was produced as a homodimeric glycoprotein with a similar molecular mass to that of the native form. Moreover, the N-terminus of the purified recombinant DSA-B protein was identical to that of the native DSA-B, confirming that the cloned cDNA encoded DSA-B. PMID:25246348

  20. Vacuolar invertases in sweet potato: molecular cloning, characterization, and analysis of gene expression.

    PubMed

    Wang, Li-Ting; Wang, Ai-Yu; Hsieh, Chang-Wen; Chen, Chih-Yu; Sung, Hsien-Yi

    2005-05-01

    Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.

  1. Characterization of grass carp (Ctenopharyngodon idella) IL-17D: molecular cloning, functional implication and signal transduction.

    PubMed

    Du, Linyong; Qin, Lei; Wang, Xinyan; Zhang, Anying; Wei, He; Zhou, Hong

    2014-02-01

    Although the roles of IL-17 family members during inflammation have been extensively studied in mammals, their knowledge in lower vertebrates is limited. In particular, the biological activities of fish IL-17 and their functional roles are largely unknown. In this study, we cloned grass carp IL-17D (gcIL-17D) and found that its putative protein possessed the conserved features of IL-17 family members. Tissue distribution analysis showed that gcIL-17D was preferentially expressed in the mucosal tissues, including skin, gill and intestine. Subsequently, the involvement of gcIL-17D in inflammatory response was demonstrated by examining the expression profiles of gcIL-17D in head kidney and head kidney leukocytes following in vivo bacterial infection and in vitro LPS treatment, respectively. Furthermore, recombinant gcIL-17D (rgcIL-17D) was prepared in grass carp kidney cells and was able to promote the gene expression of some pro-inflammatory cytokines (IL-1β, TNF-α and CXCL-8) in grass carp primary head kidney cells, revealing gcIL-17D can function as a pro-inflammatory cytokine. Moreover, rgcIL-17D appeared to activate NF-κB signaling by modulating the phosphorylation of IκBα and up-regulated CXCL-8 mRNA expression possibly through NF-κB pathway. Our data shed new light on the functional role of teleost IL-17D in inflammatory response. PMID:24120974

  2. Molecular cloning, characterization and expression of the energy homeostasis-associated gene in piglet*

    PubMed Central

    Wang, Sheng-ping; Gao, Yun-ling; Liu, Gang; Deng, Dun; Chen, Rong-jun; Zhang, Yu-zhe; Li, Li-li; Wen, Qing-qi; Hou, Yong-qing; Feng, Ze-meng; Guo, Zhao-hui

    2015-01-01

    The energy homeostasis-associated (Enho) gene encodes a secreted protein, adropin, which regulates the expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor γ, a major regulator of lipogenesis. In the present study, the porcine (Sus scrofa) homologue of the Enho gene, which was named pEnho, was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The gene sequence was submitted into the GenBank of NCBI, and the access number is GQ414763. The pEnho encodes a protein of 76 amino acids which shows 75% similarity to Homo sapiens adropin. The expression profile of pEnho in tissues (liver, muscle, anterior jejunum, posterior jejunum, and ileum) was determined by quantitative real-time RT-PCR. pEnho was localized on porcine chromosome 10 and no introns were found. In conclusion, pEnho was cloned and analysed with the aim of increasing knowledge about glucose and lipid metabolism in piglets and helping to promote the health and growth of piglets through adropin regulation. PMID:26055914

  3. Molecular cloning and functional characterization of duck mitochondrial antiviral-signaling protein (MAVS).

    PubMed

    Li, Huilin; Zhai, Yajun; Fan, Yufang; Chen, Huanchun; Zhang, Anding; Jin, Hui; Luo, Rui

    2016-03-01

    Mitochondrial antiviral-signaling protein (MAVS), also called IPS-1/VISA/Cardif, is an important molecule involved in host defense and triggers a signal for producing type I IFN. Currently the function of MAVS in ducks (duMAVS) remains largely unclear while significant progress has been made in mammals. In this study, the full-length duMAVS cDNA was cloned from duck embryo fibroblasts (DEFs) for the first time. Tissue specificity analysis showed duMAVS was universally expressed in all detected tissues. DEFs transfected with duMAVS were able to induce interferon-β (IFN-β) expression through activating interferon regulatory factor 1 (IRF1) and nuclear factor kappa B (NF-κB). Both the CARD-like domain and transmembrane domain were required for duMAVS signaling via deletion mutant analysis. In addition, poly(I:C)- or Sendai virus (SeV)-induced IFN-β expression in DEFs were significantly decreased by knock-down of duMAVS with siRNA. Altogether, these results indicate that MAVS is a critical immunoregulator in duck innate immune system.

  4. The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

    PubMed

    Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

    1992-07-01

    We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.

  5. Molecular cloning, characterization, expression and antibacterial analysis of a lysozyme homologue from Fenneropenaeus merguiensis.

    PubMed

    Mai, Wei-jun; Hu, Chao-qun

    2009-07-01

    The gene coding for lysozyme in banana prawn (Fenneropenaeus merguiensis) was cloned, sequenced and expressed in pET-32a vector. The deduced amino acid sequence of F. merguiensis lysozyme showed 37-93% similarity with the mouse, human, chicken, and tiger prawn counterparts. The lysozyme was purified to homogeneity and observed as a band of approximately 15 kDa in 12% SDS-PAGE. Semiquantitative RT-PCR analysis demonstrated that mRNA transcripts of lysozyme could be mainly detected in the tissues of hemocytes, gill, gonad and lymphoid organ of unchallenged shrimps, whereas the expression of lysozyme transcripts was increased in all the tested tissues after heat-killed Vibrio alginolyticus challenge. The temporal expression of lysozyme mRNA in hemolymph challenged by Micrococcus luteus and V. alginolyticus was both up-regulated and reached the maximum level at 8 and 16 h post stimulation, respectively, and then dropped back to the original level. Bacteriolytic activity of lysozyme against different bacterial cultures was determined by solid phase as well as turbidimetric assay. Lysis was obtained against gram positive and gram negative bacteria with strong inhibition against shrimp pathogens V. alginolyticus and V. parahemolyticus. In addition, the study of inhibition mechanism revealed that the antibacterial activity of lysozyme was a result of bactericidal effect.

  6. Cloning and molecular characterization of cationic amino acid transporter y⁺LAT1 in grass carp (Ctenopharyngodon idellus).

    PubMed

    Yang, Jixuan; Tan, Qingsong; Zhu, Wenhuan; Chen, Chen; Liang, Xufang; Pan, Lei

    2014-02-01

    The solute carrier family 7A, member 7 gene encodes the light chain- y⁺L amino acid transporter-1 (y⁺LAT1) of the heterodimeric carrier responsible for cationic amino acid (CAA) transport across the basolateral membranes of epithelial cells in intestine and kidney. Rising attention has been given to y⁺LAT1 involved in CAA metabolic pathways and growth control. The molecular characterization and function analysis of y⁺LAT1 in grass carp (Ctenopharyngodon idellus) is currently unknown. In the present study, full-length cDNA (2,688 bp), which encodes y⁺LAT1 and contains a 5'-untranslated region (319 bp), an open reading frame (1,506 bp) and a 3'-untranslated region (863 bp), has been cloned from grass carp. Amino acid sequence of grass carp y⁺LAT1 contains 11 transmembrane domains and shows 95 %, 80 % and 75 % sequence similarity to zebra fish, amphibian and mammalian y⁺LAT1, respectively. The tissue distribution and expression regulation by fasting of y⁺LAT1 mRNA were analyzed using real-time PCR. Our results showed that y⁺LAT1 mRNA was highly expressed in midgut, foregut and spleen while weakly expressed in hindgut, kidney, gill, brain, heart, liver and muscle. Nutritional status significantly influenced y⁺LAT1 mRNA expression in fish tissues, such as down-regulation of y⁺LAT1 mRNA expression after fasting (14 days).

  7. Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae.

    PubMed

    Kinsella, B T; Larkin, A; Bolton, M; Cantwell, B A

    1991-07-01

    The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed. PMID:1934116

  8. The ethics of cloning and creating embryonic stem cells as a source of tissue for transplantation: time to change the law in Australia.

    PubMed

    Savulescu, J

    2000-08-01

    Every day, people die because there are insufficient tissues available for transplantation. The development of cloning and embryonic stem (ES) cell line technologies offers real hope for developing better sources of tissues for transplantation. Moreover, these new technologies may mean that damaged tissue (for example, after a stroke or heart attack) can be replaced with normal functioning tissue rather than scar tissue. Research into 'therapeutic cloning' and the development of ES cell lines is illegal in several States in Australia. It is time to review that legislation in order to allow destructive embryo research. My argument is that at least research should be allowed on spare embryos from assisted reproduction; that it is only one moral view (of several plausible ones) of the status of the embryo which precludes producing embryos for research; that this view is mistaken and so it is morally permissible to produce embryos for research into therapeutic cloning. PMID:10985516

  9. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Colombian hospitals: dominance of a single unique multidrug-resistant clone.

    PubMed

    Gomes, A R; Sanches, I S; Aires de Sousa, M; Castañeda, E; de Lencastre, H

    2001-01-01

    The first study on the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates from Colombia was performed as part of a global surveillance established by the CEM/NET Initiative, under Project RESIST. Seventy-six MRSA isolates recovered from five hospitals during 1996-1998 were analyzed by the hybridization of ClaI restriction digests with mecA- and Tn554-specific probes, and by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests. All MRSA isolates, with one exception, belonged to a single clonal type II::NH::D. This clone, which was previously described among MRSA isolates recovered in the early 1990s in European and New York and South American hospitals, showed resistance to beta-lactam antibiotics only and appeared to be associated almost exclusively with pediatric infections ("Pediatric clone" of MRSA). While sharing identical molecular typing properties with the Pediatric clone, the Colombian isolates differed by extensive multidrug resistance and were recovered from patients of all ages. It is also noteworthy that the Brazilian clone of MRSA (XI::B::B), another multidrug-resistant international clone currently widely spread in Brazil, Argentina, Uruguay, Chile, and also in several European countries, was completely absent from this set of isolates from Colombia.

  10. Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus.

    PubMed

    Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko; Alaks, Glen; Desierto, Marie J; Townsley, Danielle M; Dumitriu, Bogdan; Chen, Jichun; Lacy, Robert C; Young, Neal S

    2015-08-15

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools. PMID:25962353

  11. Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus.

    PubMed

    Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko; Alaks, Glen; Desierto, Marie J; Townsley, Danielle M; Dumitriu, Bogdan; Chen, Jichun; Lacy, Robert C; Young, Neal S

    2015-08-15

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools.

  12. Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus

    PubMed Central

    Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko; Alaks, Glen; Desierto, Marie J; Townsley, Danielle M.; Dumitriu, Bogdan; Chen, Jichun; Lacy, Robert C.; Young, Neal S.

    2015-01-01

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools. PMID:25962353

  13. Genetic and molecular analysis of Sn, a light-inducible, tissue specific regulatory gene in maize.

    PubMed

    Tonelli, C; Consonni, G; Dolfini, S F; Dellaporta, S L; Viotti, A; Gavazzi, G

    1991-03-01

    The Sn locus of maize is functionally similar to the R and B loci, in that Sn differentially controls the tissue-specific deposition of anthocyanin pigments in certain seedling and plant cells. We show that Sn shows molecular similarity to the R gene and have used R DNA probes to characterize several Sn alleles. Northern analysis demonstrates that all Sn alleles encode a 2.5 kb transcript, which is expressed in a tissue-specific fashion consistent with the distribution of anthocyanins. Expression of the Sn gene is light-regulated. However, the Sn: bol3 allele allows Sn mRNA transcription to occur in the dark, leading to pigmentation in dark-grown seedlings and cob integuments. We report the isolation of genomic and cDNA clones of the light-independent Sn: bol3 allele. Using Sn cDNA as a probe, the spatial and temporal expression of Sn has been examined. The cell-specific localization of Sn mRNA has been confirmed by in situ hybridization using labelled antisense RNA probes. According to its proposed regulatory role, expression of Sn precedes and, in turn, causes a coordinate and tissue-specific accumulation of mRNA of structural genes for pigment synthesis and deposition, such as A1 and C2. The functional and structural relationship between R, B, Lc and Sn is discussed in terms of an evolutionary derivation from a single ancestral gene which gave rise this diverse gene family by successive duplication events.

  14. Molecular cloning and characterization of SoxB2 gene from Zhikong scallop Chlamys farreri

    NASA Astrophysics Data System (ADS)

    He, Yan; Bao, Zhenmin; Guo, Huihui; Zhang, Yueyue; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli

    2013-11-01

    The Sox proteins play critical roles during the development of animals, including sex determination and central nervous system development. In this study, the SoxB2 gene was cloned from a mollusk, the Zhikong scallop ( Chlamys farreri), and characterized with respect to phylogeny and tissue distribution. The full-length cDNA and genomic DNA sequences of C. farreri SoxB2 ( Cf SoxB2) were obtained by rapid amplification of cDNA ends and genome walking, respectively, using a partial cDNA fragment from the highly conserved DNA-binding domain, i.e., the High Mobility Group (HMG) box. The full-length cDNA sequence of Cf SoxB2 was 2 048 bp and encoded 268 amino acids protein. The genomic sequence was 5 551 bp in length with only one exon. Several conserved elements, such as the TATA-box, GC-box, CAAT-box, GATA-box, and Sox/sry-sex/testis-determining and related HMG box factors, were found in the promoter region. Furthermore, real-time quantitative reverse transcription PCR assays were carried out to assess the mRNA expression of Cf SoxB 2 in different tissues. SoxB2 was highly expressed in the mantle, moderately in the digestive gland and gill, and weakly expressed in the gonad, kidney and adductor muscle. In male and female gonads at different developmental stages of reproduction, the expression levels of Cf SoxB2 were similar. Considering the specific expression and roles of SoxB 2 in other animals, in particular vertebrates, and the fact that there are many pallial nerves in the mantle, cerebral ganglia in the digestive gland and gill nerves in gill, we propose a possible essential role in nervous tissue function for Sox B 2 in C. farreri.

  15. Molecular cloning and functional characterization of a rainbow trout liver Oatp.

    PubMed

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R

    2014-11-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772bp containing a 2115bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a Km value of 13.9μM and 13.4μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a Km value of 103μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. PMID:25218291

  16. Molecular cloning and functional characterization of a rainbow trout liver Oatp

    PubMed Central

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R.

    2014-01-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrains fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologs OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a Km value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a Km value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. PMID:25218291

  17. Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli

    PubMed Central

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Jafari, Anis; Irani, Shiva

    2015-01-01

    Background: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. Protein D (PD) belongs to the minor outer-membrane proteins of H. influenza. Moreover, it has been shown that this protein is one of the most potent vaccine candidates against the NTHi strain. Objectives: In the present study, a new truncated form of PD was designed based on conserved areas, and recombinant truncated PD was expressed. Materials and Methods: Truncated PD was designed using bioinformatics tools, and a 345 bp fragment of the truncated hpd gene was amplified by polymerase chain reaction (PCR) from H. influenzae and subsequently cloned into the prokaryotic expression vector pBAD-gIIIA. In addition, for the expression of the recombinant protein, the pBAD-truncated PD plasmid was transformed into competent TOP10 cells. The recombinant protein was expressed with Arabinose. The expressed protein was purified by affinity chromatography using Ni-NTA resin. Results: The cloning of PD was confirmed by colony-PCR and enzymatic digestion. Arabinose 0.2% was able to efficiently induce protein expression. The SDS-PAGE analysis showed that our constructed pBAD-PD-TOP10 efficiently produced a target recombinant protein with a molecular weight of 16 kDa. A high concentration of the recombinant protein was obtained via the purification process by affinity chromatography. The recombinant PD was reacted with peroxidase-conjugated rabbit anti-mouse immunoglobulins. Conclusions: Our results showed that the recombinant protein produced by the pBAD vector in the Escherichia coli system was very efficient. PMID:26464772

  18. Molecular cloning and pharmacological characterization of giant panda (Ailuropoda melanoleuca) melanocortin-4 receptor.

    PubMed

    Wang, Zhi-Qiang; Wang, Wei; Shi, Lin; Chai, Ji-Tian; Zhang, Xin-Jun; Tao, Ya-Xiong

    2016-04-01

    The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Giant panda (Ailuropoda melanoleuca), famous as the living fossil, is an endangered species endemic to China. We are interested in exploring the functions of the giant panda MC4R (amMC4R) in regulating energy homeostasis and report herein the molecular cloning and pharmacology of the amMC4R. Sequence analysis revealed that amMC4R was highly homologous (>88%) at nucleotide and amino acid sequences to several mammalian MC4Rs. Western blot revealed that the expression construct myc-amMC4R in pcDNA3.1 was successfully constructed and expressed in HEK293T cells. With human MC4R (hMC4R) as a control, pharmacological characteristics of amMC4R were analyzed with binding and signaling assays. Four agonists, including [Nle(4), D-Phe(7)]-α-melanocyte stimulating hormone (NDP-MSH), α- and β-MSH, and a small molecule agonist, THIQ, were used in binding and signaling assays. We showed that amMC4R bound NDP-MSH with the highest affinity followed by THIQ, α-MSH, and β-MSH, with the same ranking order as hMC4R. Treatment of HEK293T cells expressing amMC4R with different concentrations of agonists resulted in dose-dependent increase of intracellular cAMP levels, with similar EC50s for the four agonists. The results suggested that the cloned amMC4R encoded a functional MC4R. The availability of amMC4R and its binding and signaling properties will facilitate the investigation of amMC4R in regulating food intake and energy homeostasis.

  19. Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

    PubMed

    Ruby; Santosh Kumar, R J; Vishwakarma, Rishi K; Singh, Somesh; Khan, Bashir M

    2014-07-01

    Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.

  20. Molecular cloning and pharmacological characterization of giant panda (Ailuropoda melanoleuca) melanocortin-4 receptor.

    PubMed

    Wang, Zhi-Qiang; Wang, Wei; Shi, Lin; Chai, Ji-Tian; Zhang, Xin-Jun; Tao, Ya-Xiong

    2016-04-01

    The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Giant panda (Ailuropoda melanoleuca), famous as the living fossil, is an endangered species endemic to China. We are interested in exploring the functions of the giant panda MC4R (amMC4R) in regulating energy homeostasis and report herein the molecular cloning and pharmacology of the amMC4R. Sequence analysis revealed that amMC4R was highly homologous (>88%) at nucleotide and amino acid sequences to several mammalian MC4Rs. Western blot revealed that the expression construct myc-amMC4R in pcDNA3.1 was successfully constructed and expressed in HEK293T cells. With human MC4R (hMC4R) as a control, pharmacological characteristics of amMC4R were analyzed with binding and signaling assays. Four agonists, including [Nle(4), D-Phe(7)]-α-melanocyte stimulating hormone (NDP-MSH), α- and β-MSH, and a small molecule agonist, THIQ, were used in binding and signaling assays. We showed that amMC4R bound NDP-MSH with the highest affinity followed by THIQ, α-MSH, and β-MSH, with the same ranking order as hMC4R. Treatment of HEK293T cells expressing amMC4R with different concentrations of agonists resulted in dose-dependent increase of intracellular cAMP levels, with similar EC50s for the four agonists. The results suggested that the cloned amMC4R encoded a functional MC4R. The availability of amMC4R and its binding and signaling properties will facilitate the investigation of amMC4R in regulating food intake and energy homeostasis. PMID:26896843

  1. Molecular Cloning and Characterization of an Acetylcholinesterase cDNA in the Brown Planthopper, Nilaparvata lugens

    PubMed Central

    Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

    2010-01-01

    A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain. PMID:20874389

  2. Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut.

    PubMed

    Genta, Fernando A; Blanes, Lucas; Cristofoletti, Plínio T; do Lago, Claudimir L; Terra, Walter R; Ferreira, Clélia

    2006-10-01

    Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest k(cat)/K(M) value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane.

  3. Molecular cloning and characterization of a heat shock protein 70 gene in swimming crab (Portunus trituberculatus).

    PubMed

    Cui, Zhaoxia; Liu, Yuan; Luan, Weisha; Li, Qianqian; Wu, Danhua; Wang, Shuangyan

    2010-01-01

    Hsp70 can stimulate cells of the innate immune system directly by acting as "danger"-signaling molecules. To understand the immune defense mechanisms of swimming crab Portunus trituberculatus (Decapoda: Brachyura: Portunidae), the cDNA of Hsp70 (designated PtHsp70) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE). The full-length PtHsp70 cDNA was 2195 bp, including an open reading frame (ORF) of 1950 bp encoding a polypeptide of 650 amino acids with estimated molecular mass of 71.1514 kDa and theoretical isoelectric point of 5.38. FASTA and BLAST analysis indicated that PtHsp70 should be an inducible cytosolic member of the Hsp70 family. The coding region of PtHsp70 was uninterrupted and four SNPs with 1133C/T, 1311C/T, 1551C/T and 1809 A/G were detected by direct sequencing of 20 genomic samples. Using fluorescent real-time quantitative PCR, the transcriptional expression of PtHsp70 showed a clear time-dependent response after challenge by Vibrio alginolyticus, the main causative agent of emulsification disease causing large mortality in P. trituberculatus. This is the first report on the expression of Hsp70 induced by pathogen stimulation in Brachyura. Phylogenetic analysis revealed that the inducible Hsp70s were divided into two groups in crab and PtHsp70 was clustered into the Hsp/Hsc group (Clade I) by maximum-likelihood (ML) and Bayesian inference (BI) methods. GAP repeat and GGMP motif of inducible Hsp70 gene in the crab species were only found in Clade I. PMID:19815077

  4. Molecular cloning and characterization of a type 3 iodothyronine deiodinase in the pine snake Pituophis deppei.

    PubMed

    Villalobos, Patricia; Orozco, Aurea; Valverde-R, Carlos

    2010-11-01

    The three distinct but related isotypes of the iodothyronine deiodinase family: D1, D2, and D3, have been amply studied in vertebrate homeotherms and to a lesser extent in ectotherms, particularly in reptiles. Here, we report the molecular and kinetic characteristics of both the native and the recombinant hepatic D3 from the pine snake Pituophis deppei (PdD3). The complete PdD3 cDNA (1680 bp) encodes a protein of 287 amino acids (aa), which is the longest type 3 deiodinase so far cloned. PdD3 shares 78% identity with chicken and 71% with its other orthologs. Interestingly, the hinge domain in D3s, including PdD3, is rich in proline. This structural feature is shared with D1s, the other inner-ring deiodinases, and deserves further study. The kinetic characteristics of both native and recombinant PdD3 were similar to those reported for D3 in other vertebrates. True K(m) values for T(3) IRD were 9 and 11 nM for native and recombinant PdD3, respectively. Both exhibited a requirement for a high concentration of cofactor (40 mM DTT), insensitivity to inhibition by PTU (>2 mM), and bisubstrate, sequential-type reaction kinetics. In summary, the present data demonstrate that the liver of the adult pine snake P. deppei expresses D3. Furthermore, this is the first report of the cloning and expression of a reptilian D3 cDNA. The finding of hepatic D3 expression in the adult pine snake P. deppei is consistent with results in adult piscine species in which the dietary T(3) content seems to regulate liver deiodinase expression. Thus, our present results support the proposal that hepatic D3 in adult vertebrates plays a sentinel role in avoiding an inappropriate overload of exogenous T(3) secondary to feeding in those species that devour the whole prey.

  5. Molecular cloning and characterization of wheat calreticulin (CRT) gene involved in drought-stressed responses.

    PubMed

    Jia, Xiao-Yun; Xu, Chong-Yi; Jing, Rui-Lian; Li, Run-Zhi; Mao, Xin-Guo; Wang, Ji-Ping; Chang, Xiao-Ping

    2008-01-01

    Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca(2+)-binding protein in multicellular eukaryotes. CRT plays a crucial role in many cellular processes including Ca(2+) storage and release, protein synthesis, and molecular chaperone activity. To elucidate the function of CRTs in plant responses against drought, a main abiotic stress limiting cereal crop production worldwide, a full-length cDNA encoding calreticulin protein namely TaCRT was isolated from wheat (Triticum aestivum L.). The deduced amino acid sequence of TaCRT shares high homology with other plant CRTs. Phylogenetic analysis indicates that TaCRT cDNA clone encodes a wheat CRT3 isoform. Southern analysis suggests that the wheat genome contains three copies of TaCRT. Subcellular locations of TaCRT were the cytoplasm and nucleus, evidenced by transient expression of GFP fused with TaCRT in onion epidermal cells. Enhanced accumulation of TaCRT transcript was observed in wheat seedlings in response to PEG-induced drought stress. To investigate further whether TaCRT is involved in the drought-stress response, transgenic plants were constructed. Compared to the wild-type and GFP-expressing plants, TaCRT-overexpressing tobacco (Nicotiana benthamiana) plants grew better and exhibited less wilt under the drought stress. Moreover, TaCRT-overexpressing plants exhibited enhanced drought resistance to water deficit, as shown by their capacity to maintain higher WUE (water use efficiency), WRA (water retention ability), RWC (relative water content), and lower MDR (membrane damaging ratio) (P < or = 0.01) under water-stress conditions. In conclusion, a cDNA clone encoding wheat CRT was successfully isolated and the results suggest that TaCRT is involved in the plant response to drought stress, indicating a potential in the transgenic improvements of plant water-stress.

  6. Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

    PubMed

    Ruby; Santosh Kumar, R J; Vishwakarma, Rishi K; Singh, Somesh; Khan, Bashir M

    2014-07-01

    Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism. PMID:24664316

  7. Molecular cloning and functional characterization of a rainbow trout liver Oatp

    SciTech Connect

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R.

    2014-11-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K{sub m} value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K{sub m} value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. - Highlights: • A new Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. • rtOatp1d1 is predominantly expressed in the liver. • rtOatp1d1 displays multi-specific transport of endogenous and xenobiotic substrates. • rtOatp1d1 is a homologue of the OATP1A1, OATP1B1 and OATP1B3. • rtOatp1d1 is a microcystin (MC) transporter.

  8. Molecular cloning of a cDNA encoding a novel fatty acid-binding protein from rat skin.

    PubMed

    Watanabe, R; Fujii, H; Odani, S; Sakakibara, J; Yamamoto, A; Ito, M; Ono, T

    1994-04-15

    A novel skin-type fatty acid-binding protein, termed cutaneous(C)-FABP, has been purified from rat skin and a cDNA clone for this protein has been identified. The purified protein had the ability to bind long chain fatty acids like other rat FABPs. The deduced amino acid sequence of the cDNA clone comprises residues yielding a molecular mass for the polypeptide of 15.1 kDa and exhibits around 50% identity to myelin P2 protein, adipocyte FABP and heart FABP. Our results propose that C-FABP is a new member of the FABP family.

  9. Molecular cloning of the recA gene and construction of a recA strain of Francisella novicida.

    PubMed Central

    Berg, J M; Mdluli, K E; Nano, F E

    1992-01-01

    A gene locus that is functionally analogous to the recA gene of Escherichia coli was molecularly cloned from Francisella novicida. The cloned gene was found to suppress the sensitivity of an E. coli strain to DNA-damaging agents and to support genetic recombination in E. coli. After transposon mutagenesis, the recA-like gene locus was returned to F. novicida and a UV-sensitive F. novicida strain was isolated. In contrast to the wild-type strain, this UV-sensitive strain could not be transformed with chromosomal DNA. Images PMID:1309722

  10. Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3.

    PubMed

    Kolakowski, L F; O'Neill, G P; Howard, A D; Broussard, S R; Sullivan, K A; Feighner, S D; Sawzdargo, M; Nguyen, T; Kargman, S; Shiao, L L; Hreniuk, D L; Tan, C P; Evans, J; Abramovitz, M; Chateauneuf, A; Coulombe, N; Ng, G; Johnson, M P; Tharian, A; Khoshbouei, H; George, S R; Smith, R G; O'Dowd, B F

    1998-12-01

    Galanin is a 29- or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K(D) = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively.

  11. [Soft tissue tumors - the view of the molecular biologist].

    PubMed

    Krsková, Lenka; Mrhalová, Marcela; Kalinová, Markéta; Campr, Vít; Capková, Linda; Grega, Marek; Háček, Jaromír; Hornofová, Ludmila; Chadimová, Mária; Chmelová, Renata; Kodetová, Daniela; Zámečník, Josef; Kodet, Roman

    2014-07-01

    Soft tissue tumors (SSTs) constitute a broad spectrum of neoplasms with diverse biological properties. Rare or unusual types are often difficult to classify. Recent studies show, that a significant subset of SSTs including many types of sarcomas are associated with specific genetic changes such as chromosomal translocations producing chimeric genes, which play a role in the pathogenesis of SSTs. Because SSTs represent a diagnostically challenging group of tumors, molecular-genetic techniques (FISH or PCR) are useful as supplementary and/or confirmatory diagnostic tools. In the present paper we demonstrate the usefulness of a combined diagnostic approach using the tools of classical histopathology and immunohistochemistry together with the molecular diagnostic approach in selected nosologic entites. PMID:25186594

  12. Construction and characterization of HIV type 1 CRF07_BC infectious molecular clone from men who have sex with men.

    PubMed

    Jiang, Yan-Ling; Bai, Wen-Wei; Qu, Fan-Wei; Ma, Hua; Jiang, Run-Sheng; Shen, Bao-Sheng

    2016-03-01

    This study aimed to investigate the biological characterization of HIV type 1 (HIV-1) CRF07_BC infection among men who have sex with men (MSM). From November 2011 to November 2013, a total of 66 blood samples were collected from MSM with acute HIV-1 infection with CRF07_BC subgroup strains. Deletion in the gag p6 region was detected by sequence alignment and comparative analysis. Peripheral blood mononuclear cells (PBMCs) of HNXX1301-1307 samples were separated by density gradient centrifugation. Nested polymerase chain reaction (nPCR) was used to amplify the viral DNA. The near full-length HIV-1 DNA products were ligated to the long terminal repeat (LTR) vector plasmid (07BCLTR) to construct a full-length HIV clone. The molecular clone was transfected into HEK-293T cells, TZM-b1 cells and patients' PBMCs. The pregenome of an infectious molecular clone of HIV-1 (pNL4-3) was amplified, and a subclone with CRF07_BC was developed to construct the full-length chimeric molecular clone pNL4-3/07BCLTR. Detection of p24 antigen and luciferase activity was used to measure the in vitro infectivity of pNL4-3/07BCLTR. Among the 66 MSM patients infected with CRF07_BC strains, deletion mutations of the Gag P6 proteins were found in 7 of 18CRF07_BC strains; deletion mutations of 2-13 amino acids in different regions were discovered in 6 strains; and the remaining 42 strains did not show deletions. Seven strains with amino acids deficiency in the P6 protein accounted for 27% of all strains and 75% of all deletion genotype strains. A total of 186 full-length molecular clones of CRF07_BC were constructed. There were 5, 9, 10 and 11 clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 that resulted in p24-positive supernatant when transfected into HEK-293T cells. Full-length clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 showed slight infection in the transfected TZM-b1 cells, as judged by the fluorescence values of TZM-b1 cells 48h post-transfection. However, we were unable to

  13. Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23).

    PubMed

    Alvarez-Pérez, Marco Antonio; Narayanan, Sampath; Zeichner-David, Margarita; Rodríguez Carmona, Bruno; Arzate, Higinio

    2006-03-01

    Cementum is a unique mineralized connective tissue that covers the root surfaces of the teeth. The cementum is critical for appropriate maturation of the periodontium, both during development as well as that associated with regeneration of periodontal tissues, IU; however, one major impediment to understand the molecular mechanisms that regulate periodontal regeneration is the lack of cementum markers. Here we report on the identification and characterization of one such differentially human expressed gene, termed "cementum protein-23" (CP-23) that appears to be periodontal ligament and cementum-specific. We screened human cementum tumor-derived cDNA libraries by transient expression in COS-7 cells and "panning" with a rabbit polyclonal antibody against a cementoblastoma conditioned media-derived protein (CP). One isolated cDNA, CP-23, was expressed in E. coli and polyclonal antibodies against the recombinant human CP-23 were produced. Expression of CP-23 protein by cells of the periodontium was examined by Northern blot and in situ hybridization. Expression of CP-23 transcripts in human cementoblastoma-derived cells, periodontal ligament cells, human gingival fibroblasts and alveolar bone-derived cells was determined by RT-PCR. Our results show that we have isolated a 1374-bp human cDNA containing an open reading frame that encodes a polypeptide with 247 amino acid residues, with a predicted molecular mass of 25.9 kDa that represents CP species. The recombinant human CP-23 protein cross-reacted with antibodies against CP and type X collagen. Immunoscreening of human periodontal tissues revealed that CP-23 gene product is localized to the cementoid matrix of cementum and cementoblasts throughout the entire surface of the root, cell subpopulations of the periodontal ligament as well as cells located paravascularly to the blood vessels into the periodontal ligament. Furthermore, 98% of putative cementoblasts and 15% of periodontal ligament cells cultured in vitro

  14. Molecular cloning and functional analysis of zebrafish (Danio rerio) chemokine genes.

    PubMed

    Chen, Li-Chen; Chen, Jyh-Yih; Hour, Ai-Ling; Shiau, Chyuan-Yuan; Hui, Cho-Fat; Wu, Jen-Leih

    2008-12-01

    Chemokines control leukocyte trafficking which plays important roles in resistance to pathogenic infection. Five CXC chemokines have been reported in the zebrafish (Danio rerio) in GenBank, and herein we named them CXC-46, -56, -64, -66, and scyba. Through RT-PCR for cloning and sequencing these chemokines, the cDNA sequences of CXC-46, -56, -64, and -66 of zebrafish were determined, and it was found that the cDNA sequences were the same as those published in GenBank. Phylogenetic analysis revealed that zebrafish scyba is closest to the CXCL14 subgroup, CXC-46 is closest to the human CCL25 and catfish CXCL-2-like gene, and CXC-56, -64, and -66 are closest to the catfish CXCL10 subgroup. Further study of the tissue-specific, lipopolysaccharide (LPS) stimulation-specific, and polyinosinic-polycytidylic acid (poly I:C) stimulation-specific expressions of these five zebrafish CXC chemokine messenger (m)RNAs were determined by a comparative reverse-transcription polymerase chain reaction (RT-PCR). The RT-PCR revealed a high level of constitutive expression of CXC-56 in many tissues including the eyes, fins, heart, liver, muscles, and skin. Starvation had significant effects on the gene expressions of several zebrafish CXC chemokines including CXC-56, -64, -66, and scyba compared to the control group. Zebrafish CXC chemokines showed a concave pattern of expression after stimulation with LPS. Following poly I:C treatment of between 0.1 and 10 g/fish, dose-dependent effects were revealed. Temperature and acid-base conditions affected these zebrafish chemokines by increasing their induction compared to the control group, except for CXC-64 which exhibited no significant differences in either condition. Furthermore, these novel research results indicate that chemokines can be markers of different experimental conditions. PMID:18778789

  15. Molecular cloning, expression and characterization of programmed cell death 10 from sheep (Ovis aries).

    PubMed

    Yang, Yong-Jie; Liu, Zeng-Shan; Lu, Shi-Ying; Li, Chuang; Hu, Pan; Li, Yan-Song; Liu, Nan-Nan; Tang, Feng; Xu, Yun-Ming; Zhang, Jun-Hui; Li, Zhao-Hui; Feng, Xiao-Li; Zhou, Yu; Ren, Hong-Lin

    2015-03-01

    Programmed cell death 10 (PDCD10) is a highly conserved adaptor protein. Its mutations result in cerebral cavernous malformations (CCMs). In this study, PDCD10 cDNA from the buffy coat of Small Tail Han sheep (Ovis aries) was cloned from a suppression subtractive hybridization cDNA library, named OaPDCD10. The full-length cDNA of OaPDCD10 was 1343bp with a 639bp open reading frame (ORF) encoding 212 amino acid residues. Tissue distribution of OaPDCD10 mRNA determined that it was ubiquitously expressed in all tested tissue samples, and the highest expression was observed in the heart. The differential expression of OaPDCD10 between infected sheep (challenged with Brucella melitensis) and vaccinated sheep (vaccinated with Brucella suis S2) was also investigated. The results revealed that, compared to the control group, the expression of OaPDCD10 from infected and vaccinated sheep was both significantly up-regulated (p<0.05). Moreover, the expression levels of OaPDCD10 from the vaccinated sheep were significantly higher than the infected sheep (p<0.05) after 30days post-inoculation. The recombinant OaPDCD10 (rOaPDCD10) protein was expressed in Escherichia coli BL21 (DE3), and then purified by affinity chromatography. The rOaPDCD10 protein was demonstrated to induce apoptosis and promote cell proliferation. Our studies are intended to discover potential diagnostic biomarkers of brucellosis to discern infected sheep from vaccinated sheep, and OaPDCD10 could be considered as a potential diagnostic biomarker of brucellosis.

  16. Molecular cloning and characterization of three beta-defensins from canine testes.

    PubMed

    Sang, Yongming; Ortega, M Teresa; Blecha, Frank; Prakash, Om; Melgarejo, Tonatiuh

    2005-05-01

    Mammalian beta-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine beta-defensin isoforms (cBD-1, -2, and -3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the beta-defensin consensus six-cysteine motif. The two longer isoforms, cBD-2 and -3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine beta-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus; MICs of 6 and 100 mug/ml, respectively), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Neisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 mug/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 mug/ml) and lower activity against Ureaplasma urealyticum and U. canigenitalium (MIC of 200 mug/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine beta-defensins were highly expressed in testis. In situ hybridization indicated that cBD-1 was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast, cBD-2 was located primarily within Leydig cells. The longest isoform, cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine beta-defensins play an important role in host defense and other physiological functions of the male reproductive system. PMID:15845463

  17. Molecular cloning, characterization and expression of cathepsin D from grass carp (Ctenopharyngodon idella).

    PubMed

    Dong, Zhong-dian; Zhang, Jiao; Ji, Xiang-shan; Zhou, Fen-na; Fu, Yong; Chen, Weiyun; Zeng, Yong-qing; Li, Tong-ming; Wang, Hui

    2012-11-01

    Cathepsin D is a lysosomal aspartic proteinase which participates in various degradation functions within the cell. In this current study, we cloned and characterized the complete cDNA of grass carp cathepsin D through 5'- and 3'-RACE. The cathepsin D contained a 56 bp 5' terminal untranslated region (5'-UTR), a 1197 bp open reading frame encoding 398 amino acids, and a 394 bp 3'-UTR. Grass carp cathepsin D shared high similarity with those from other species, and showed the highest amino acid identity of 91% to Danio rerio. Unlike many other organisms, the grass carp cathepsin D contains only one N-glycosylation site closest to the N-terminal. Real-time quantitative RT-PCR demonstrated that Cathepsin D expressed in all twelve tissues (bladder, brain, liver, heart, gill, muscle, fin, eye, intestines, spleen, gonad and head kidney). The relative expression levels of Cathepsin D in gonad and liver were 26.58 and 24.95 times as much as those in fin, respectively. The expression level of Cathepsin D in muscle approximately 16-fold higher, in intestines and spleen were 12-fold higher. The cathepsin D expression showed an upward trend during embryonic development. After challenged with Aeromonas hydrophil, the expression of grass carp cathepsin D gene showed significant changes in the four test tissues (liver, head kidney, spleen and intestines). The fact that the bacterial infection can obviously improve the cathepsin D expression in immune-related organs, may suggest that cathepsin D plays an important role in the innate immune response of grass carp.

  18. Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus.

    PubMed

    Hull, J Joe; Wang, Meixian

    2014-01-01

    The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits. PMID:26463065

  19. Molecular cloning of the mouse CCK gene: expression in different brain regions and during cortical development.

    PubMed Central

    Vitale, M; Vashishtha, A; Linzer, E; Powell, D J; Friedman, J M

    1991-01-01

    In this paper we describe experiments that address specific issues concerning the regulation of the mouse cholecystokinin gene in brain and intestine. The mouse cholecystokinin gene was cloned and sequenced. Extensive homology among the mouse, man and rat genes was noted particularly in the three exons and the regions upstream of the RNA start site. RNAse protection assays for each of the three exons were used to demonstrate that CCK is expressed in only a subset of tissues and that the same cap site and splice choices are used in brain, intestine as well as in cerebellum, cortex, midbrain, hypothalamus and hippocampus. CCK RNA was also noted to be detectable in kidney. Thus the same gene using the same promoter is expressed in subsets of cells that differ in their biochemical, morphologic and functional characteristics. The level of expression of CCK was also monitored during mouse cortical development and the appearance of CCK RNA was compared to glutamate decarboxylase (GAD), enkephalin and somatostatin. It was noted that each of these cortical markers was first expressed at different times during cortical development. The appearance of CCK RNA during intestinal development was also measured and found to precede appearance in cortex by several days. Images PMID:2011497

  20. The molecular cloning and expression of two CRABP cDNAs from human skin

    SciTech Connect

    Eller, M.S.; Oleksiak, M.F.; McQuaid, T.J.; McAfee, S.G.; Gilchrest, B.A. Boston Univ., MA )

    1992-02-01

    Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). The authors have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABPI cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. They conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. The data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.

  1. Molecular cloning, characterization and expression analysis of the complement component C6 gene in grass carp.

    PubMed

    Shen, Yu-Bang; Zhang, Jun-Bin; Xu, Xiao-Yan; Li, Jia-Le

    2011-05-15

    The complement system, as a representative of innate immunity, plays a key role in the host defense against infections. C6 is the member of complement components creating the membrane attack complex (MAC). In this study, we cloned and characterized the grass carp complement component C6 (gcC6) gene. Our data showed that gcC6 gene contained a 2724bp open reading frame (ORF), a 237bp 5'-untranslated region (UTR) and a 219bp 3'-UTR. The deduced amino acid sequence of gcC6 showed 77.6% and 58.9% identity to zebrafish C6 and rainbow trout C6, respectively. GcC6 gene was expressed in a wide range of grass carp tissues, and the highest expression level of gcC6 was detected in the spleen and liver. Upon challenge with Aeromonas hydrophila, its expression was significantly up-regulated in muscle, trunk kidney, liver, head kidney, spleen, heart and intestine, whereas it was down-regulated in the brain and skin. The expression level of gcC6 was high at the unfertilized egg stage. It was significantly increased at 1 day post-hatching, but it was decreased at 10 days post-hatching. This result suggested that the complement C6 transcripts in early embryos were of maternal origin. PMID:21353312

  2. Molecular cloning and characterization of lymphocyte cell kinase from humphead snapper (Lutjanus sanguineus).

    PubMed

    Huang, Y; Cai, J; Wang, B; Tang, J-F; Jian, J-C; Wu, Z-H; Gan, Z; Lu, Y-S

    2016-07-01

    Lymphocyte cell kinase (LCK) belongs to the Src family of tyrosine kinases, which involves in the proliferation control of lymphocytes. In this study, we cloned the LCK gene of humphead snapper (Lutjanus sanguineus) (designed as LsLCK). Sequence analysis showed that the full-length cDNA of LsLCK was 2279 bp, contained a 1506-bp open reading frame (ORF), encoding a polypeptide of 501 amino acids. The deduced amino acid possessed the typical structural features of known LCK proteins, including four Src homology (SH) domains arranged as the SH1 domain followed by a regulatory C-terminal tail (COOH-domain), SH2 and SH3 adapter domains and SH4 domain which required for membrane attachment and CD4/CD8 binding. Fluorescent quantitative real-time PCR analysis indicated that LsLCK transcripts were expressed mainly in thymus, spleen and head kidney in healthy fish. Moreover, the mRNA expressions in these tissues were significantly up-regulated after challenge with Vibrio harveyi. The results of immunohistochemistry showed that LsLCK protein localized distinctly in cytoplasm of cell in thymus, spleen and head kidney. Taken together, these findings indicated that LsLCK may play an important role in the immune response of humphead snapper against bacterial infection.

  3. Molecular cloning of bullfrog saxiphilin: a unique relative of the transferrin family that binds saxitoxin.

    PubMed Central

    Morabito, M A; Moczydlowski, E

    1994-01-01

    Plasma and tissue of certain vertebrates contain a protein called saxiphilin that specifically binds the neurotoxin saxitoxin with nanomolar affinity. We describe the isolation of a cDNA clone of saxiphilin from liver of the North American bullfrog (Rana catesbeiana). The cDNA sequence encodes a protein that is evolutionarily related to members of the transferrin family of Fe(3+)-binding proteins. Pairwise sequence alignment of saxiphilin with various transferrins reveals amino acid identity as high as 51% and predicts 14 disulfide bonds that are highly conserved. The larger size of saxiphilin (91 kDa) versus serum transferrin (approximately 78 kDa) is primarily due to a unique insertion of 144 residues. This insertion contains a 49-residue domain classified as a type 1 repetitive element of thyroglobulin, which is shared by a variety of membrane, secreted, and extracellular matrix proteins. Saxiphilin also differs from transferrins in 9 of 10 highly conserved amino acids in the two homologous Fe3+/HCO3-binding sites of transferrin. Identification of saxiphilin implies that transferrin-like proteins comprise a diverse superfamily with functions other than iron binding. Images PMID:8146142

  4. Molecular cloning and functional characterization of spexin in orange-spotted grouper (Epinephelus coioides).

    PubMed

    Li, Shuisheng; Liu, Qiongyu; Xiao, Ling; Chen, Huapu; Li, Guangli; Zhang, Yong; Lin, Haoran

    2016-01-01

    Spexin is a newly discovered neuropeptide in vertebrates. Comprehensive comparative studies are required to unveil its biological functions. In order to ascertain the neuroendocrine function of spexin in orange-spotted grouper, its full-length cDNA and genomic DNA sequences were cloned and analyzed. Sequence analyses showed that the spexin gene structure is composed of six exons and five introns, and the amino acids of mature peptide (spexin-14) in grouper are identical to that of other fish. Tissue expression analysis found that grouper spexin is highly expressed in the brain, liver and ovary. Real time-PCR analysis demonstrated that the hypothalamic expression of spexin declined gradually during the ovarian development, and was up-regulated by food deprivation. Intraperitoneal administration of spexin-14 peptides to grouper significantly elevated the mRNA levels of proopiomelanocortin (pomc) and suppressed the orexin expression in the hypothalamus, but could not change the hypothalamic expression of gonadotropin releasing hormone 1 (gnrh1). Both in vivo and in vitro administration of spexin could not significantly influence the expression of follicle-stimulating hormone β (fshβ) and luteinizing hormone β (lhβ) in the pituitary with the exception of an inhibition of gh expression. Our data suggested that the spexin has a significant role in the regulation of energy metabolism and food intake in orange-spotted grouper. PMID:26944307

  5. Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

    PubMed

    Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

    2016-01-01

    Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding. PMID:27173207

  6. Molecular cloning and characterisation of the griffon vulture (Gyps fulvus) toll-like receptor 1.

    PubMed

    de la Lastra, José Manuel Pérez; de la Fuente, José

    2007-01-01

    The toll-like receptor (TLR) family is an ancient pattern recognition receptor family, conserved from insects to mammals. Members of the TLR family are vital to immune function through the sensing of pathogenic agents and initiation of an appropriate immune response. In this study, we cloned a cDNA encoding for a griffon vulture (Gyps fulvus) orthologue of mammalian TLR1 (CD281). The predicted 650 amino acid sequence comprised an extracellular domain with five leucine-rich repeats (LRR) and an LRR-C-terminal (LRR-CT) motif, followed by a 23 amino acid transmembrane segment, and a 190 amino acid intracytoplasmic region containing the Toll/IL-1R (TIR) domain. Vulture TLR1 and TIR domain showed 64% and 86% amino acid sequence similarity with chicken sequences. The tissue and cell expression pattern of vulture TLR1 were analysed by real time-PCR (RT-PCR) and correlated with the ability to respond to various pathogenic challenges. Despite the similarities in the overall structure and expression pattern of vulture TLR1 with other vertebrate TLRs, the length of the vulture TLR ectodomain, number and position of LRRs and N-glycosylation sites suggest structural differences that may have functional implications.

  7. Molecular cloning, functional expression, and chromosomal localization of mouse hepatocyte nuclear factor 1.

    PubMed Central

    Kuo, C J; Conley, P B; Hsieh, C L; Francke, U; Crabtree, G R

    1990-01-01

    The homeodomain-containing transcription factor hepatocyte nuclear factor 1 (HNF-1) most likely plays an essential role during liver organogenesis by transactivating a family of greater than 15 predominantly hepatic genes. We have isolated cDNA clones encoding mouse HNF-1 and expressed them in monkey COS cells and in the human T-cell line Jurkat, producing HNF-1 DNA-binding activity as well as transactivation of reporter constructs containing multimerized HNF-1 binding sites. In addition, the HNF-1 gene was assigned by somatic cell hybrids and recombinant inbred strain mapping to mouse chromosome 5 near Bcd-1 and to human chromosome 12 region q22-qter, revealing a homologous chromosome region in these two species. The presence of HNF-1 mRNA in multiple endodermal tissues (liver, stomach, intestine) suggests that HNF-1 may constitute an early marker for endodermal, rather than hepatocyte, differentiation. Further, that HNF-1 DNA-binding and transcriptional activity can be conferred by transfecting the HNF-1 cDNA into several cell lines indicates that it is sufficient to activate transcription in the context of ubiquitously expressed factors. Images PMID:2263635

  8. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.

    PubMed Central

    Plowman, G D; Whitney, G S; Neubauer, M G; Green, J M; McDonald, V L; Todaro, G J; Shoyab, M

    1990-01-01

    Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth. Images PMID:2164210

  9. Molecular cloning and characterization of lymphocyte cell kinase from humphead snapper (Lutjanus sanguineus).

    PubMed

    Huang, Y; Cai, J; Wang, B; Tang, J-F; Jian, J-C; Wu, Z-H; Gan, Z; Lu, Y-S

    2016-07-01

    Lymphocyte cell kinase (LCK) belongs to the Src family of tyrosine kinases, which involves in the proliferation control of lymphocytes. In this study, we cloned the LCK gene of humphead snapper (Lutjanus sanguineus) (designed as LsLCK). Sequence analysis showed that the full-length cDNA of LsLCK was 2279 bp, contained a 1506-bp open reading frame (ORF), encoding a polypeptide of 501 amino acids. The deduced amino acid possessed the typical structural features of known LCK proteins, including four Src homology (SH) domains arranged as the SH1 domain followed by a regulatory C-terminal tail (COOH-domain), SH2 and SH3 adapter domains and SH4 domain which required for membrane attachment and CD4/CD8 binding. Fluorescent quantitative real-time PCR analysis indicated that LsLCK transcripts were expressed mainly in thymus, spleen and head kidney in healthy fish. Moreover, the mRNA expressions in these tissues were significantly up-regulated after challenge with Vibrio harveyi. The results of immunohistochemistry showed that LsLCK protein localized distinctly in cytoplasm of cell in thymus, spleen and head kidney. Taken together, these findings indicated that LsLCK may play an important role in the immune response of humphead snapper against bacterial infection. PMID:26660470

  10. Molecular Cloning, Recombinant Expression, and Funtional Characterization of APRIL (TNFSF13) in Cat (felis catus).

    PubMed

    Liu, Hong-Zhen; Song, Ren; Zhang, Jia-Xin; Li, Jian-Feng; Gu, Wei; Zhang, Shuang-Quan

    2016-01-01

    A proliferation-inducing ligand (APRIL) is a critical member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the cDNA of cat APRIL (cAPRIL) was successfully amplified. Sequence analysis showed that the open reading frame (ORF) of cAPRIL contains a putative furin protease cleavage site (R-R-K-R), a conserved putative N-glycosylation site (Asn(124)), and two conservative cysteine residues (Cys(196) and Cys(211)). Real-time quantitative PCR (qPCR) analysis revealed that cAPRIL could be detected in various tissues. The phylogenetic analysis and predicted three dimensional (3D) structure revealed that it is similar to its counterparts. The extracellular soluble domain of the cAPRIL (csAPRIL) fragment was cloned into the expression vector pET43.1a. SDS-PAGE and Western blotting analysis indicated a high-level expression of csAPRIL protein in Escherichia coli BL21 (DE3). MTT assays revealed that purified recombinant csAPRIL protein was able to stimulate proliferation of mouse B-cells. These findings indicate that cAPRIL plays an important role in proliferation of B-cells and provide the basis for investigation on the roles of APRIL in this important domestic species. PMID:26485397

  11. Molecular cloning, expression, and evolution analysis of type II CHI gene from peanut (Arachis hypogaea L.).

    PubMed

    Liu, Yu; Zhao, Shuzhen; Wang, Jiangshan; Zhao, Chuanzhi; Guan, Hongshan; Hou, Lei; Li, Changsheng; Xia, Han; Wang, Xingjun

    2015-01-01

    Chalcone isomerase (CHI) plays critical roles in plant secondary metabolism, which is important for the interaction between plants and the environment. CHI genes are widely studied in various higher plants. However, little information about CHI genes is available in peanut. Based on conservation of CHI gene family, we cloned the peanut type II CHI gene (AhCHI II) cDNA and genome sequence. The amino acid sequence of peanut CHI II was highly homologous to type II CHI from other plant species. qRT-PCR results showed that peanut CHI II is mainly expressed in roots; however, peanut CHI I is mainly expressed in tissues with high content of anthocyanin. Gene duplication and gene cluster analysis indicated that CHI II was derived from CHI I 65 million years ago approximately. Our gene structure analysis results are not in agreement with the previous hypothesis that CHI II was derived from CHI I by the insertion of an intron into the first exon. Moreover, no positive selection pressure was found in CHIs, while, 32.1 % of sites were under neutral selection, which may lead to mutation accumulation and fixation during great changes of environment.

  12. Molecular cloning and characterization of orange-spotted grouper (Epinephelus coioides) CXC chemokine ligand 12.

    PubMed

    Wu, Chen-Shiou; Wang, Ting-Yu; Liu, Chin-Feng; Lin, Hao-Ping; Chen, Young-Mao; Chen, Tzong-Yueh

    2015-12-01

    Chemokines are a family of soluble peptides that can recruit a wide range of immune cells to sites of infection and disease. The CXCL12 is a chemokine that binds to its cognate receptor CXCR4 and thus involved in multiple physiological and pathophysiological processes. In this study, we cloned and characterized CXCL12 from Epinephelus coioides (osgCXCL12). We found that the open reading frame of osgCXCL12 consists of 98 amino acid residues with the small cytokine C-X-C domain located between residues 29 and 87. Higher expression levels for osgCXCL12 were detected at the kitting stage, compared with the prolarva and larva shape stages. The expression patterns revealed that osgCXCL12 may play a key role in early grouper development. We detected mRNA transcripts for osgCXCL12 in healthy tissues and found the highest osgCXCL12 expression in the head kidney. Furthermore, a time-course analysis revealed significantly increased osgCXCL12 and osgCXCR4 expression levels after the nervous necrosis virus (NNV) challenge. In addition, expression of osgCXCL12 was affected by injection with microbial mimics [LPS and poly(I:C)]. These results suggest that osgCXCL12 is associated with inflammatory and developmental processes in the grouper.

  13. Molecular cloning and functional characterization of spexin in orange-spotted grouper (Epinephelus coioides).

    PubMed

    Li, Shuisheng; Liu, Qiongyu; Xiao, Ling; Chen, Huapu; Li, Guangli; Zhang, Yong; Lin, Haoran

    2016-01-01

    Spexin is a newly discovered neuropeptide in vertebrates. Comprehensive comparative studies are required to unveil its biological functions. In order to ascertain the neuroendocrine function of spexin in orange-spotted grouper, its full-length cDNA and genomic DNA sequences were cloned and analyzed. Sequence analyses showed that the spexin gene structure is composed of six exons and five introns, and the amino acids of mature peptide (spexin-14) in grouper are identical to that of other fish. Tissue expression analysis found that grouper spexin is highly expressed in the brain, liver and ovary. Real time-PCR analysis demonstrated that the hypothalamic expression of spexin declined gradually during the ovarian development, and was up-regulated by food deprivation. Intraperitoneal administration of spexin-14 peptides to grouper significantly elevated the mRNA levels of proopiomelanocortin (pomc) and suppressed the orexin expression in the hypothalamus, but could not change the hypothalamic expression of gonadotropin releasing hormone 1 (gnrh1). Both in vivo and in vitro administration of spexin could not significantly influence the expression of follicle-stimulating hormone β (fshβ) and luteinizing hormone β (lhβ) in the pituitary with the exception of an inhibition of gh expression. Our data suggested that the spexin has a significant role in the regulation of energy metabolism and food intake in orange-spotted grouper.

  14. Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus

    PubMed Central

    Hull, J. Joe; Wang, Meixian

    2014-01-01

    The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits. PMID:26463065

  15. Molecular cloning of allelopathy related genes and their relation to HHO in Eupatorium adenophorum.

    PubMed

    Guo, Huiming; Pei, Xixiang; Wan, Fanghao; Cheng, Hongmei

    2011-10-01

    In this study, conserved sequence regions of HMGR, DXR, and CHS (encoding 3-hydroxy-3-methylglutaryl-CoA reductase, 1-deoxyxylulose-5-phosphate reductoisomerase and chalcone synthase, respectively) were amplified by reverse transcriptase (RT)-PCR from Eupatorium adenophorum. Quantitative real-time PCR showed that the expression of CHS was related to the level of HHO, an allelochemical isolated from E. adenophorum. Semi-quantitative RT-PCR showed that there was no significant difference in expression of genes among three different tissues, except for CHS. Southern blotting indicated that at least three CHS genes are present in the E. adenophorum genome. A full-length cDNA from CHS genes (named EaCHS1, GenBank ID: FJ913888) was cloned. The 1,455 bp cDNA contained an open reading frame (1,206 bp) encoding a protein of 401 amino acids. Preliminary bioinformatics analysis of EaCHS1 revealed that EaCHS1 was a member of CHS family, the subcellular localization predicted that EaCHS1 was a cytoplasmic protein. To the best of our knowledge, this is the first report of conserved sequences of these genes and of a full-length EaCHS1 gene in E. adenophorum. The results indicated that CHS gene is related to allelopathy of E. adenophorum.

  16. Molecular Cloning and Characterization of Violaxanthin De-Epoxidase (CsVDE) in Cucumber

    PubMed Central

    Huang, Hongyu; Kong, Lingcui; Niu, Dandan; Sui, Xiaolei; Zhang, Zhenxian

    2013-01-01

    Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under high light stress. We have cloned a violaxanthin de-epoxidase gene (CsVDE) from cucumber. The amino acid sequence of CsVDE has high homology with VDEs in other plants. RT-PCR analysis and histochemical staining show that CsVDE is expressed in all green tissues in cucumber and Arabidopsis. Using GFP fusion protein and immunogold labeling methods, we show that CsVDE is mainly localized in chloroplasts in cucumber. Under high light stress, relative expression of CsVDE and the de-epoxidation ratio (A+Z)/(V+A+Z) is increased rapidly, and abundance of the gold particles was also increased. Furthermore, CsVDE is quickly induced by cold and drought stress, reaching maximum levels at the 2nd hour and the 9th day, respectively. The ratio of (A+Z)/(V+A+Z) and non-photochemical quenching (NPQ) is reduced in transgenic Arabidopsis down-regulated by the antisense fragment of CsVDE, compared to wild type (WT) Arabidopsis under high light stress. This indicates decreased functionality of the xanthophyll cycle and increased sensitivity to photoinhibition of photosystem II (PSII) in transgenic Arabidopsis under high light stress. PMID:23717606

  17. Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio.

    PubMed

    Qi, Lin; Xiang, Zhiming

    2015-07-01

    The death domain-associated protein Daxx exerts many functions including the induction and inhibition of apoptosis, regulation of chromatin remodeling and gene transcription. In this report, we have cloned and characterized a Daxx ortholog from the zebrafish, Danio rerio. The bioinformatics analysis results indicated that the open reading frame (ORF) of zebrafish Daxx is 2,151bp long and encodes a putative protein of 716 amino acids containing Daxx domain. Though quantitative PCR analyses, Daxx mRNA was detected in embryonic development from 6 h to 120 h and in all 11 selected zebrafish tissues, and the expression of Daxx was increased first and then decreased during megalocytivirus infectious spleen and kidney necrosis virus (ISKNV) infection. Fluorescence microscopy indicated that the full-length protein was located in the nuclei of the tested Hela cells uniformly but punctiform distribution in HEK293T. In the luciferase report assays, the GAL4-Daxx fusion protein inhibited the transcriptional activity of L8G5-Luc reporter gene showed that Daxx might act as a transcriptional repressor, following the over-expression in HEK293T, the activation of NF-κB-Luc and p53/p21-Luc reporter genes were repressed by the protein. These results suggested that Daxx might play definite role in apoptosis and innate immunity in zebrafish.

  18. Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

    PubMed

    Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

    2016-01-01

    Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding.

  19. Molecular cloning, characterization, and sexually dimorphic expression of five major sex differentiation-related genes in a Scorpaeniform fish, sablefish (Anoplopoma fimbria).

    PubMed

    Smith, Elizabeth K; Guzmán, José M; Luckenbach, J Adam

    2013-06-01

    Regardless of how sex is determined, the gonadal genes expressed downstream that regulate sex differentiation are relatively conserved among vertebrates. The goal of this study was to clone and characterize five key sex differentiation-related genes in a Scorpaeniform fish, sablefish (Anoplopoma fimbria). Complete mRNA sequences of foxl2, cyp19a1a, dmrt1, sox9a and amh were cloned, sequenced, and phylogenetically analyzed. The sablefish mRNA sequences exhibited the characteristic domains of each gene. The deduced amino sequences were highly conserved in some cases, such as Foxl2, whereas others, such as Amh, exhibited lower homology to corresponding sequences in other vertebrates. Using quantitative PCRs developed for each gene, we found that foxl2 and cyp19a1a mRNA levels were significantly elevated in juvenile sablefish ovaries compared to testes, whereas dmrt1, sox9a and amh mRNA levels were significantly elevated in testes relative to ovaries. These patterns were upheld in our tissue distribution analyses of adult fish, but overall four of the genes, foxl2, cyp19a1a, dmrt1 and amh, were robust markers of sex in sablefish. This study provides important molecular tools for ongoing work related to sex control in sablefish and exploration of the earliest period of molecular sex differentiation and its regulation. PMID:23507626

  20. Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.

    PubMed Central

    De Luca, V; Marineau, C; Brisson, N

    1989-01-01

    The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the alpha-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases. Images PMID:2704736

  1. Unveiling aminopeptidase P from Streptomyces lavendulae: molecular cloning, expression and biochemical characterization.

    PubMed

    Nandan, Arya S; Nampoothiri, Kesavan Madhavan

    2014-02-01

    Presence of proline residues in the second position of the N-terminus in peptides restricts the usage of many aminopeptidases; however, aminopeptidase P (APP) is capable of removing this blockage. Based on the N-terminal amino acid sequences of APP from Streptomyces lavendulae, app gene was cloned in pET28a(+) and over expressed as a His-tagged protein with a molecular weight of ≈60 kDa in Escherichia coli BL21 (DE3). Nucleotide sequencing revealed a 1467 bp open reading frame encoding 488 amino acids (NCBI Accession No: GenBank: KC292272.1). The substrate specificity of the recombinant APP was analyzed by the hydrolysis of the Xaa-Pro bond in Gly-Pro dipeptide and bradykinin. K(m) and V(max) of the enzyme were found to be 0.4697 mmol l⁻¹ and 0.6396 μmol min⁻¹, respectively. APP activity was enhanced in the presence of metal ions such as Co²⁺, Mn²⁺, Mg²⁺ and Cu²⁺ ions and was inhibited by 1,10-phenanthroline, EDTA, PMSF and DTT. The atomic absorption studies revealed the presence of Mn²⁺ in the protein as a co-factor. This substrate specific metalloenzyme was found to be a tetramer and optimally active at pH 8 and 37 °C.

  2. Molecular cloning and evolutionary analysis of the GJA1 (connexin43) gene from bats (Chiroptera).

    PubMed

    Wang, Li; Li, Gang; Wang, Jinhong; Ye, Shaohui; Jones, Gareth; Zhang, Shuyi

    2009-04-01

    Gap junction protein connexin43 (Cx43), encoded by the GJA1 gene, is the most abundant connexin in the cardiovascular system and was reported as a crucial factor maintaining cardiac electrical conduction, as well as having a very important function in facilitating the recycling of potassium ions from hair cells in the cochlea back into the cochlear endolymph during auditory transduction processes. In mammals, bats are the only taxon possessing powered flight, placing exceptional demand on many organismal processes. To meet the demands of flying, the hearts of bats show many specialties. Moreover, ultrasonic echolocation allows bat species to orientate and often detect and locate food in darkness. In this study, we cloned the full-length coding region of GJA1 gene from 12 different species of bats and obtained orthologous sequences from other mammals. We used the maximum likelihood method to analyse the evolution of GJA1 gene in mammals and the lineage of bats. Our results showed this gene is much conserved in mammals, as well as in bats' lineage. Compared with other mammals, we found one private amino acid substitution shared by bats, which is located on the inner loop domain, as well as some species-specific amino acid substitutions. The evolution rate analyses showed the signature of purifying selection on not only different classification level lineages but also the different domains and amino acid residue sites of this gene. Also, we suggested that GJA1 gene could be used as a good molecular marker to do the phylogenetic reconstruction.

  3. Molecular cloning and characterization of a chlorophyll degradation regulatory gene (ZjSGR) from Zoysia japonica.

    PubMed

    Teng, K; Chang, Z H; Xiao, G Z; Guo, W E; Xu, L X; Chao, Y H; Han, L B

    2016-01-01

    The stay-green gene (SGR) is a key regulatory factor for chlorophyll degradation and senescence. However, to date, little is known about SGR in Zoysia japonica. In this study, ZjSGR was cloned, using rapid amplification of cDNA ends-polymerase chain reaction (PCR). The target sequence is 831 bp in length, corresponding to 276 amino acids. Protein BLAST results showed that ZjSGR belongs to the stay-green superfamily. A phylogenetic analysis implied that ZjSGR is most closely related to ZmSGR1. The subcellular localization of ZjSGR was investigated, using an Agrobacterium-mediated transient expression assay in Nicotiana benthamiana. Our results demonstrated that ZjSGR protein is localized in the chloroplasts. Quantitative real time PCR was carried out to investigate the expression characteristics of ZjSGR. The expression level of ZjSGR was found to be highest in leaves, and could be strongly induced by natural senescence, darkness, abscisic acid (ABA), and methyl jasmonate treatment. Moreover, an in vivo function analysis indicated that transient overexpression of ZjSGR could accelerate chlorophyll degradation, up-regulate the expression of SAG113, and activate ABA biosynthesis. Taken together, these results provide evidence that ZjSGR could play an important regulatory role in leaf chlorophyll degradation and senescence in plants at the molecular level. PMID:27173268

  4. Molecular cloning and characterization of a tropinone reductase from Dendrobium nobile Lindl.

    PubMed

    Chen, Wei; Cheng, Xiaofei; Zhou, Zhenhua; Liu, Junjun; Wang, Huizhong

    2013-02-01

    A cDNA sequence that encodes a peptide with similarity to known tropinone reductases (TR) was cloned from Dendrobium nobile Lindl. The full coding region of the gene (DnTR1) is 804 bp in length which encodes a putative peptide consisting of 268 amino acids. Phylogenetic analysis showed that DnTR1 was a novel member of the TR family and evolutionarily distant from those well-characterized subgroups of TRs, suggesting that DnTR1 may have distinct characteristics. Structural modeling found that DnTR1 had a similar electrostatic environment at the inner molecular surface of the substrate binding pocket with TRI encoded by Datura stramonium (DsTRI). Catalytic activity assay with recombinant protein demonstrated that DnTR1 was able to reduce tropinone, 3-quinuclidinone hydrochloride, and 4-methylcyclohexanone using NADPH as coenzyme. Gene expression profiling by qRT-PCR revealed that the DnTR1 transcript was expressed in all three vegetative organs (leaves, stems and roots) of D. nobile with the highest expression level in roots. The expression of DnTR1 mRNA was enhanced 9.5 times (P < 0.01) by treatment of methyl jasmonate at 24 h, but not affected by salicylic acid and sodium nitroprusside treatments, indicating that DnTR1 regulation may be involved in a jasmonate-dependent pathway. PMID:23104472

  5. Simple and versatile molecular method of copy-number measurement using cloned competitors.

    PubMed

    Kim, Hyun-Kyoung; Hwang, Hai-Li; Park, Seong-Yeol; Lee, Kwang Man; Park, Won Cheol; Kim, Han-Seong; Um, Tae-Hyun; Hong, Young Jun; Lee, Jin Kyung; Joo, Sun-Young; Seoh, Ju-Young; Song, Yeong-Wook; Kim, Soo-Youl; Kim, Yong-Nyun; Hong, Kyeong-Man

    2013-01-01

    Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.

  6. Molecular cloning, overexpression and characterization of a novel feruloyl esterase from a soil metagenomic library.

    PubMed

    Sang, Shu Li; Li, Gang; Hu, Xiao Peng; Liu, Yu Huan

    2011-01-01

    The gene estF27, encoding a protein with feruloyl esterase activity, was cloned through functional screening from a soil metagenomic library and expressed in Escherichiacoli BL21 (DE3) with high solubility. Sequence analysis showed that estF27 encoded a protein of 291 amino acids with a predicted molecular mass of 31.16 kDa. According to the substrate specificity, EstF27 was classified as a type A feruloyl esterase. EstF27 displayed optimal activity at 40°C and pH 6.8. This enzyme was stable in a broad pH range of 5.0-10.0 over 24 h, and retained more than 50% of its activity after 96 or 120 h incubation in the presence of 3 M KCl or 5 M NaCl. The enzyme activity was slightly enhanced by the addition of Mg(2+) and Fe(3+) at a low concentration, and completely inhibited by Cu(2+). In the enzymatic hydrolysis of destarched wheat bran, EstF27 could release ferulic acid from it in the presence of xylanase from Thermomyces lanuginosus. Given its alkalitolerance, halotolerance and highly soluble expression, EstF27 is a promising candidate for industrial applications.

  7. Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6.

    PubMed

    Zhu, Yanbing; Zheng, Wenguang; Ni, Hui; Liu, Han; Xiao, Anfeng; Cai, Huinong

    2015-10-01

    A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.

  8. Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima).

    PubMed

    Ruiz-Sanchez, Alejandro; Cruz-Camarillo, Ramon; Salcedo-Hernandez, Ruben; Ibarra, Jorge E; Barboza-Corona, Jose Eleazar

    2005-10-01

    An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5alphaF', and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5'-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0-10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55 degrees C.

  9. Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

    PubMed Central

    Kim, Hyun-Kyoung; Hwang, Hai-Li; Park, Seong-Yeol; Lee, Kwang Man; Park, Won Cheol; Kim, Han-Seong; Um, Tae-Hyun; Hong, Young Jun; Lee, Jin Kyung; Joo, Sun-Young; Seoh, Ju-Young; Song, Yeong-Wook; Kim, Soo-Youl; Kim, Yong-Nyun; Hong, Kyeong-Man

    2013-01-01

    Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes. PMID:23936009

  10. Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae.

    PubMed

    Satake, Ryoko; Ichiyanagi, Atsushi; Ichikawa, Keiichi; Hirokawa, Kozo; Araki, Yasuko; Yoshimura, Taro; Gomi, Keiko

    2015-11-01

    Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels.

  11. Sea bass ghrelin: molecular cloning and mRNA quantification during fasting and refeeding.

    PubMed

    Terova, Genciana; Rimoldi, Simona; Bernardini, Giovanni; Gornati, Rosalba; Saroglia, Marco

    2008-01-15

    Ghrelin is a novel appetite-inducing peptide hormone secreted by the stomach. The purpose of this study was first to identify the cDNA encoding sequence for ghrelin in sea bass (Dicentrarchus labrax). Using molecular cloning techniques we sequenced the cDNA corresponding to sea bass ghrelin mRNA. A total of 798 bases including a 5'-untranslated region (89 bp), an open reading frame (ORF) (324 bp), and a 3'-untranslated region (385 bp) were detected. Nucleotide sequence (ORF) encoded a 108 amino acid prepropeptide that demonstrated complete conservation of the N-terminal "biological active core" (GSSF) of the predicted mature ghrelin peptide. We also analyzed fasting-induced changes in the expression of ghrelin mRNA, using a one-tube two-temperature real-time RT-PCR with which the gene expression can be absolutely quantified using the standard curve method. Our results revealed that ghrelin was highly expressed in the stomach with much lower levels of expression in the proximal intestine and brain. Levels of ghrelin mRNA in the stomach were upregulated under conditions of negative energy balance, such as starvation, and downregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass ghrelin gene and support a role of this orexigenic hormone in the regulation of food intake in sea bass.

  12. Characterization and molecular cloning of a serine hydroxymethyltransferase 1 (OsSHM1) in rice.

    PubMed

    Wang, Dekai; Liu, Heqin; Li, Sujuan; Zhai, Guowei; Shao, Jianfeng; Tao, Yuezhi

    2015-09-01

    Serine hydroxymethyltransferase (SHMT) is important for one carbon metabolism and photorespiration in higher plants for its participation in plant growth and development, and resistance to biotic and abiotic stresses. A rice serine hydroxymethyltransferase gene, OsSHM1, an ortholog of Arabidopsis SHM1, was isolated using map-based cloning. The osshm1 mutant had chlorotic lesions and a considerably smaller, lethal phenotype under natural ambient CO2 concentrations, but could be restored to wild type with normal growth under elevated CO2 levels (0.5% CO2 ), showing a typical photorespiratory phenotype. The data from antioxidant enzymes activity measurement suggested that osshm1 was subjected to significant oxidative stress. Also, OsSHM1 was expressed in all organs tested (root, culm, leaf, and young panicle) but predominantly in leaves. OsSHM1 protein is localized to the mitochondria. Our study suggested that molecular function of the OsSHM1 gene is conserved in rice and Arabidopsis. PMID:25641188

  13. Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato.

    PubMed Central

    du Jardin, P; Rojas-Beltran, J; Gebhardt, C; Brasseur, R

    1995-01-01

    A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe. PMID:8552717

  14. Molecular cloning and characterization of amylase from soil metagenomic library derived from Northwestern Himalayas.

    PubMed

    Sharma, Sarika; Khan, Farrah Gul; Qazi, Ghulam Nabi

    2010-05-01

    The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase, protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as substrates. The maximal activity (2.46 U/mg) was observed at 40 degrees C under nearly neutral pH conditions with amylose; whereas it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division.

  15. Molecular cloning, phylogenetic analysis and expression of beluga whale (Delphinapterus leucas) interleukin 6.

    PubMed

    St-Laurent, G; Archambault, D

    2000-01-31

    Interleukin 6 (IL-6) is a cytokine produced primarily by the monocytes/macrophages with regulatory effects in hematopoiesis, acute phase response, and multiple aspects of the immune response. IL-6 exerts its activity through its binding to specific high affinity receptors at the surface of target cells. As yet, no molecular data have been reported for the beluga whale IL-6. In this study, we cloned and determined the entire beluga whale IL-6-encoding cDNA sequence by reverse transcription-polymerase chain reaction (RT-PCR) sequencing, and analysed its genetic relationship with those from several mammalian species including human, rodent, ruminant, carnivore and other marine species. The identity levels of beluga whale IL-6 nucleic and deduced amino acid sequences with those from these mammalian species ranged from 62.3 to 97.3%, and 42.9 to 95.6%, respectively. Phylogenetic analysis based on amino acid sequences showed that the beluga whale IL-6 was most closely related to that of the killer whale. Thereafter, beluga whale IL-6-encoding sequence was successfully expressed in Escherichia coli by using the pTHIOHisA expression vector for the production of a recombinant fusion protein. The immunogenicity of the recombinant fusion protein was then confirmed as determined by the production of a beluga whale IL-6-specific rabbit antiserum.

  16. Molecular imaging of biological tissue using gas cluster ions

    PubMed Central

    Tian, Hua; Wucher, Andreas; Winograd, Nicholas

    2015-01-01

    An Arn+ (n = 1–6000) gas cluster ion source has been utilized to map the chemical distribution of lipids in a mouse brain tissue section. We also show that the signal from high mass species can be further enhanced by doping a small amount of CH4 into the Ar cluster to enhance the ionization of several biologically important molecules. Coupled with secondary ion mass spectrometry instrumentation which utilizes a continuous Ar cluster ion projectile, maximum spatial resolution and maximum mass resolution can be achieved at the same time. With this arrangement, it is possible to achieve chemically resolved molecular ion images at the 4-µm resolution level. The focused Arn+/[Arx(CH4)y]+ beams (4–10 µm) have been applied to the study of untreated mouse brain tissue. A high signal level of molecular ions and salt adducts, mainly from various phosphocholine lipids, has been seen and directly used to map the chemical distribution. The signal intensity obtained using the pure Ar cluster source, the CH4-doped cluster source and C60 is also presented. PMID:26207076

  17. Molecular cloning and expression analysis of cytochrome c oxidase subunit II from Sitophilus zeamais.

    PubMed

    Hou, Chang-Liang; Wang, Jing-Bo; Wu, Hua; Liu, Jia-Yu; Ma, Zhi-Qing; Feng, Jun-Tao; Zhang, Xing

    2016-09-30

    Cytochrome c oxidase subunit II (COX II) containing a dual core CuA active site is one of the core subunits of mitochondrial Cytochrome c oxidase (Cco), which plays a significant role in the physiological process. In this report, the full-length cDNA of COXII gene was cloned from Sitophilus zeamais, which had an open reading frame (ORF) of 684 bp encoding 227 amino acids residues. The predicted COXII protein had a molecular mass of 26.2 kDa with pI value of 6.37. multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXII had high sequence identity with the COXII of other insect species. The gene was subcloned into the expression vector pET-32a, and induced by isopropyl β-d-thiogalactopyranoside (IPTG) in E. coli Transetta (DE3) expression system. Finally the recombinant COXII with 6-His tag was purified using affinity chromatography with Ni(2+)-NTA agarose. Western Blotting (WB) showed the recombinant protein was about 44 kD, and the concentration of fusion protein was 50 μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXII could catalyze the oxidation of substrate Cytochrome C (Cyt c), and influenced by allyl isothiocyanate (AITC). By using molecular docking method, It was found that a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results suggested that tag-free COXII was functional and one of the action sites of AITC, which will be helpful to carry out a point mutation in binding sites for the future research. PMID:27614312

  18. Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis.

    PubMed Central

    Manca, C; Lyashchenko, K; Wiker, H G; Usai, D; Colangeli, R; Gennaro, M L

    1997-01-01

    Proteins that are actively secreted by Mycobacterium tuberculosis generate immune responses in the infected host. This has prompted the characterization of protein components of mycobacterial culture filtrates to develop subunit vaccines and immunodiagnostic reagents. Fractionation of filtrates of M. tuberculosis cultures has yielded an abundant protein called MPT63, which has an apparent molecular mass of 18 kDa. We report the molecular cloning and nucleotide sequence of the mpt63 gene, purification of recombinant MPT63 antigen from Escherichia coli cells, and serological characterization of MPT63. Nucleotide sequence analysis of mpt63 identified an open reading frame encoding a protein of 159 amino acids (aa) consisting of a 29-aa secretion signal peptide and a 130-aa mature MPT63 protein. Recombinant MPT63 protein, purified from E. coli cells, and native MPT63, purified from M. tuberculosis culture filtrates, were indistinguishable in serological assays. Thus, the recombinant protein constitutes a valuable reagent for immunological studies. MPT63 evoked humoral immune responses in guinea pigs infected with virulent M. tuberculosis by the aerosol route. The mpt63 gene is found only in species of the M. tuberculosis complex, as shown by DNA hybridization experiments. Moreover, polyclonal antibody against MPT63 does not cross-react with proteins of a common environmental mycobacterial species, Mycobacterium avium. The absence of cross-reactive epitopes makes MPT63 an attractive candidate as an M. tuberculosis complex-specific diagnostic reagent. In particular, evaluation of MPT63 as an M. tuberculosis complex-specific reagent for diagnostic skin testing is under way. PMID:8975887

  19. Molecular Cloning and Expression of Pro J 1: A New Allergen of Prosopis Juliflora Pollen.

    PubMed

    Dousti, Fatemeh; Assarehzadegan, Mohammad-Ali; Morakabati, Payam; Khosravi, Gholam Reza; Akbari, Bahareh

    2016-04-01

    Pollen from mesquite (Prosopis juliflora) is one of the important causes of immediate hypersensitivity reactions in the arid and semi-arid regions of the world. The aim of present study is to produce and purify the recombinant form of allergenic Ole e 1-like protein from the pollen of this allergenic tree. Immunological and cross-inhibition assays were performed for the evaluation of IgE-binding capacity of purified recombinant protein. For molecular cloning, the coding sequence of the mesquite Ole e 1-like protein was inserted into pTZ57R/T vector and expressed in Escherichia coli using the vector pET-21b(+). After purification of the recombinant protein, its immunoreactivity was analysed by in vitro assays using sera from twenty one patients with an allergy to mesquite pollen. The purified recombinant allergen was a member of Ole e 1-like protein family and consisted of 150 amino acid residues, with a predicted molecular mass of 16.5 kDa and a calculated isoelectric point (pI) of 4.75. Twelve patients (57.14%) had significant specific IgE levels for this recombinant allergen. Immunodetection and inhibition assays indicated that the purified recombinant allergen might be the same as that in the crude extract. Herein, we introduce an important new allergen from P. juliflora pollen (Pro j 1), which is a member of the Ole e 1-like protein family and exhibits significant identity and similarity to other allergenic members of this family.

  20. Molecular cloning, characterization, and expression of Cuc m 2, a major allergen in Cucumis melo

    PubMed Central

    Sankian, Mojtaba; Mahmoudi, Mahmoud; Varasteh, Abdol-Reza

    2013-01-01

    Background: Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon. Methods: Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli. Results: Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5’and 3’ UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins. Conclusion: Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins. PMID:26989709

  1. Molecular and structural preservation of dehydrated bio-tissue for THz spectroscopy

    NASA Astrophysics Data System (ADS)

    Png, Gretel M.; Choi, Jin Wook; Guest, Ian; Ng, Brian W.-H.; Mickan, Samuel P.; Abbott, Derek; Zhang, Xi-Cheng

    2007-12-01

    Terahertz transmission through freshly excised biological tissue is limited by the tissue's high water content. Tissue fixation methods that remove water, such as fixation in Formalin, destroy the structural information of proteins hence are not suitable for THz applications. Dehydration is one possible method for revealing the tissue's underlying molecular structure and components. In this study, we measured the THz responses over time of dehydrating fresh, necrotic and lyophilized rat tissue. Our results show that as expected, THz absorption increases dramatically with drying and tissue freshness can be maintained through lyophilization. Dehydrated biological tissue with retained molecular structure can be useful for future laser-based THz wave molecular analysis.

  2. Molecular cloning, expression pattern, and 3D structural prediction of the cold inducible RNA-binding protein (CIRP) in Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Yang, Xiao; Gao, Jinning; Ma, Liman; Li, Zan; Wang, Wenji; Wang, Zhongkai; Yu, Haiyang; Qi, Jie; Wang, Xubo; Wang, Zhigang; Zhang, Quanqi

    2015-02-01

    Cold-inducible RNA-binding protein (CIRP) is a kind of RNA binding proteins that plays important roles in many physiological processes. The CIRP has been widely studied in mammals and amphibians since it was first cloned from mammals. On the contrary, there are little reports in teleosts. In this study, the Po CIRP gene of the Japanese flounder was cloned and sequenced. The genomic sequence consists of seven exons and six introns. The putative PoCIRP protein of flounder was 198 amino acid residues long containing the RNA recognition motif (RRM). Phylogenetic analysis showed that the flounder PoCIRP is highly conserved with other teleost CIRPs. The 5' flanking sequence was cloned by genome walking and many transcription factor binding sites were identified. There is a CpGs region located in promoter and exon I region and the methylation state is low. Quantitative real-time PCR analysis uncovered that Po CIRP gene was widely expressed in adult tissues with the highest expression level in the ovary. The mRNA of the Po CIRP was maternally deposited and the expression level of the gene was regulated up during the gastrula and neurula stages. In order to gain the information how the protein interacts with mRNA, we performed the modeling of the 3D structure of the flounder PoCIRP. The results showed a cleft existing the surface of the molecular. Taken together, the results indicate that the CIRP is a multifunctional molecular in teleosts and the findings about the structure provide valuable information for understanding the basis of this protein's function.

  3. Molecular cloning and functional expression of a human intestinal lactoferrin receptor.

    PubMed

    Suzuki, Y A; Shin, K; Lönnerdal, B

    2001-12-25

    Lactoferrin (Lf), a major iron-binding protein in human milk, has been suggested to have multiple biological roles such as facilitating iron absorption, modulating the immune system, embryonic development, and cell proliferation. Our previous binding studies suggested the presence of a specific receptor for Lf (LfR) in the small intestine of newborn infants, which may facilitate iron absorption. We here report the cloning and the functional expression of the human intestinal LfR and the evidence of its involvement in iron metabolism. The entire coding region of the LfR cDNA was cloned by PCR based on amino acid sequences of the purified native LfR (nLfR). The recombinant LfR (rLfR) was then expressed in a baculovirus-insect cell system and purified by immobilized human Lf (hLf) affinity chromatography where binding of hLf to the rLfR was partially Ca(2+) dependent. The apparent molecular mass was 136 kDa under nonreducing conditions and 34 kDa under reducing conditions. 125I-hLf bound to the rLfR with an apparent K(d) of approximately 360 nM. These biochemical properties of the rLfR are similar to those of the nLfR. RT-PCR revealed that the gene was expressed at high levels in fetal small intestine and in adult heart and at lower levels in Caco-2 cells. PI-PLC treatment of Caco-2 cells indicated that the LfR is GPI anchored. In Caco-2 cells transfected with the LfR gene, 125I-hLf binding and 59Fe-hLf uptake were increased by 1.7 and 3.4 times, respectively, compared to those in mock-transfected cells. Our findings demonstrate the presence of a unique receptor-mediated mechanism for nutrient uptake by the newborn.

  4. Molecular cloning, mRNA expression, and characterization of HSP90 gene from Chinese mitten crab Eriocheir japonica sinensis.

    PubMed

    Li, Peng; Zha, Jie; Zhang, Zhenhua; Huang, Hua; Sun, Hongying; Song, Daxiang; Zhou, Kaiya

    2009-07-01

    HSP90 is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. Using expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques, an HSP90 gene designated as EjsHSP90 was cloned and characterized from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsHSP90 is 2,517 bp and contains an open reading frame of 2157 bp which encodes a 718 amino acid polypeptide (82.8 kDa) bearing characteristics of the HSP90 family and an ATP binding domain. Sequence alignment shows that EjsHSP90 shared 79%-96% identity with HSP90 sequences reported in other animals, and it shares identical structural features. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsHSP90 mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that EjsHSP90 is expressed throughout the three developmental stages but expression levels varied among different body parts of crabs. EjsHSP90 mRNA expression in the abdomen of the first crab stage is consistently higher than that of the other two stages, suggesting that EjsHSP90 gene is involved in the crabs' early developmental process, especially in the crab brachyurization process. Results from quantitative RT-PCR excluded the possibility that the expression of EjsHSP90 mRNA is induced primarily by osmotic stress. Phylogenetic analyses reveal that HSP90 gene is informative and complementary for reconstruction of arthropod phylogenetic relationships. PMID:19166961

  5. Molecular cloning and functional characterization of a novel i-type lysozyme in the freshwater mussel Cristaria plicata.

    PubMed

    Dai, Wenjuan; Wu, Dan; Zhang, Ming; Wen, Chungen; Xie, Yanhai; Hu, Baoqing; Jian, Shaoqing; Zeng, Mingyu; Tao, Zhiying

    2015-12-01

    The freshwater bivalve Cristaria plicata, which is widely distributed in Eastern Asia, is a key species in the pearl culture industry. In this study, a novel invertebrate-type lysozyme, designated as CpLYZ2, was cloned from hemocytes of C. plicata. This lysozyme shares high sequence identity and is homologous to a previously identified lysozyme CpLYZ1 isolated from C. plicata and with HcLyso3 isolated from Hyriopsis cumingii. The full-length cDNA of CpLYZ2 is 913 bp long, which includes an open reading frame (ORF) of 486 bp, a 3' untranslated region (UTR) of 389 bp and a 5' UTR of 38 bp. The ORF encodes a putative polypeptide of 161 amino acids with a predicted molecular mass of 18.2 kDa and a theoretical isoelectric point of 6.56. CpLYZ2 mRNA transcripts can be detected in hemocytes, hepatopancreas, muscle, gills and mantle tissues, the greatest expression being observed in the gills. CpLYZ2 expression in hemocytes, hepatopancreas and gills increased significantly after the mussel was challenged with Aeromonas hydrophila. Furthermore, the optimal pH and temperature for enzyme activity of the recombinant CpLYZ2 were 5.5 and 50°C, respectively. The recombinant lysozyme protein exhibited bacteriolytic activity against Escherichia coli, A. hydrophila, Staphylococcus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis. The findings of this study help to elucidate immune responses in molluscs and will thus expedite disease management of these key freshwater species, in turn boosting pearl culture in eastern Asia. PMID:26589461

  6. Mining tissue-specific contigs from peanut (Arachis hypogaea L.) for promoter cloning by deep transcriptome sequencing.

    PubMed

    Geng, Lili; Duan, Xiaohong; Liang, Chun; Shu, Changlong; Song, Fuping; Zhang, Jie

    2014-10-01

    Peanut (Arachis hypogaea L.), one of the most important oil legumes in the world, is heavily damaged by white grubs. Tissue-specific promoters are needed to incorporate insect resistance genes into peanut by genetic transformation to control the subterranean pests. Transcriptome sequencing is the most effective way to analyze differential gene expression in this non-model species and contribute to promoter cloning. The transcriptomes of the roots, seeds and leaves of peanut were sequenced using Illumina technology. A simple digital expression profile was established based on number of transcripts per million clean tags (TPM) from different tissues. Subsequently, 584 root-specific candidate transcript assembly contigs (TACs) and 316 seed-specific candidate TACs were identified. Among these candidate TACs, 55.3% were root-specific and 64.6% were seed-specific by semi-quantitative RT-PCR analysis. Moreover, the consistency of semi-quantitative RT-PCR with the simple digital expression profile was correlated with the length and TPM value of TACs. The results of gene ontology showed that some root-specific TACs are involved in stress resistance and respond to auxin stimulus, whereas, seed-specific candidate TACs are involved in embryo development, lipid storage and long-chain fatty acid biosynthesis. One root-specific promoter was cloned and characterized. We developed a high-yield screening system in peanut by establishing a simple digital expression profile based on Illumina sequencing. The feasible and rapid method presented by this study can be used for other non-model crops to explore tissue-specific or spatially specific promoters.

  7. Mining tissue-specific contigs from peanut (Arachis hypogaea L.) for promoter cloning by deep transcriptome sequencing.

    PubMed

    Geng, Lili; Duan, Xiaohong; Liang, Chun; Shu, Changlong; Song, Fuping; Zhang, Jie

    2014-10-01

    Peanut (Arachis hypogaea L.), one of the most important oil legumes in the world, is heavily damaged by white grubs. Tissue-specific promoters are needed to incorporate insect resistance genes into peanut by genetic transformation to control the subterranean pests. Transcriptome sequencing is the most effective way to analyze differential gene expression in this non-model species and contribute to promoter cloning. The transcriptomes of the roots, seeds and leaves of peanut were sequenced using Illumina technology. A simple digital expression profile was established based on number of transcripts per million clean tags (TPM) from different tissues. Subsequently, 584 root-specific candidate transcript assembly contigs (TACs) and 316 seed-specific candidate TACs were identified. Among these candidate TACs, 55.3% were root-specific and 64.6% were seed-specific by semi-quantitative RT-PCR analysis. Moreover, the consistency of semi-quantitative RT-PCR with the simple digital expression profile was correlated with the length and TPM value of TACs. The results of gene ontology showed that some root-specific TACs are involved in stress resistance and respond to auxin stimulus, whereas, seed-specific candidate TACs are involved in embryo development, lipid storage and long-chain fatty acid biosynthesis. One root-specific promoter was cloned and characterized. We developed a high-yield screening system in peanut by establishing a simple digital expression profile based on Illumina sequencing. The feasible and rapid method presented by this study can be used for other non-model crops to explore tissue-specific or spatially specific promoters. PMID:25231965

  8. Generation of a molecular clone of an attenuated lentivirus, a first step in understanding cytopathogenicity and virulence.

    PubMed

    Blatti-Cardinaux, Laure; Pisoni, Giuliano; Stoffel, Michael H; Zanoni, Reto; Zahno, Marie-Luise; Bertoni, Giuseppe

    2016-01-01

    Small ruminant lentiviruses infect goats and sheep, inducing clinical disease in a minority of infected animals. Following an eradication campaign, clinical cases may disappear in a population. The complete elimination of these lentiviruses is however difficult to achieve and the spreading of less virulent strains often parallels the elimination of their virulent counterparts. Here, we characterized three such strains isolated from a flock in the post-eradication phase. We completely sequenced their genomes, showing that one of the isolates was most probably the product of a recombination event between the other two viruses. By comparing the sequences of these isolates with those of virulent strains, we found evidence that particular LTR mutations may explain their attenuated phenotype. Finally, we constructed an infectious molecular clone representative of these viruses, analyzing its replication characteristics in different target cells. This clone will permit us to explore the molecular correlates of cytopathogenicity and virulence.

  9. Molecular cloning of covalently closed circular DNA of bovine leukemia virus.

    PubMed Central

    Kashmiri, S V; Mehdi, R; Ferrer, J F

    1984-01-01

    The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs. Images PMID:6319758

  10. Identification and cloning of molecular markers for UV-B tolerant gene in wild sugarcane (Saccharum spontaneum L.).

    PubMed

    Li, Yuan; He, Yongmei; Zu, Yanqun; Zhan, Fangdong

    2011-11-01

    Previously we have selected wild sugarcane (Saccharum spontaneum L.) sterile lines that are tolerant or susceptible to UV-B radiation based on response index (RI) in a field screening test. The RI was established according to plant height, tiller number, leaf index, total biomass and brix under enhanced ultraviolet-B (UV-B, 280-310 nm) radiation. In this experiment, molecular markers linked to the UV-B tolerant and susceptible genes were identified and cloned. RAPD (Randomly amplified polymorphic DNAs) assay using 100 arbitrary primers followed by clustering analysis separated the tolerant and susceptible lines into two groups at the genetic distance of 0.380. The UV-B tolerant and susceptible gene pools were constructed and compared using the Bulked Segregate Analysis (BSA) approach. Of the 100 arbitrary RAPD primers, primer OPR16 produced polymorphic DNA banding patterns from both gene pools. The OPR16-1200 bp DNA fragment was only amplified from the tolerant lines and the OPR16-800 bp from the susceptible ones. These two PCR fragments were cloned onto T-vector. DNA sequence alignment analysis determined that 42% homology existed between the reverse and forward sequences of the OPR16-1200 bp clone, and 36% homology between the forward sequences of the OPR16-800 bp and OPR16-1200 bp clones. The two DNA clones were determined to be linked to the UV-B tolerant and susceptible genes, and they can be used to develop molecular markers for the associated traits.

  11. Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus

    PubMed Central

    Park, Eun-Mi; Kang, Jung-Ha; Seo, Jung Soo; Kim, GunDo; Chung, Jongkyeong; Choi, Tae-Jin

    2008-01-01

    Background Signal transducer and activator of transcription 1 (STAT1) is a critical component of interferon (IFN)-alpha/beta and IFN-gamma signaling. Although seven isoforms of STAT proteins have been reported from mammals, limited information is available for the STAT genes in fish. We isolated complementary DNA with high similarity to mammalian STAT1 from the olive flounder, Paralichthys olivaceus. Results A DNA fragment containing the conserved SH2 domain was amplified by RT-PCR using degenerate primers designed based on the highly conserved sequences in the SH2 domains of the zebrafish and mammalian STAT1. The complete cDNA sequence was obtained by 5' and 3' RACE. The flounder STAT1 transcript consisted of 2,909 bp that encoded a polypeptide of 749 amino acids. The overall similarity between flounder STAT1 and other STATs was very high, with the highest amino acid sequence identity to snakehead (89%). Phylogenetic analyses reveal that flounder STAT1 is in the same monophyletic group with snakehead STAT1. Quantitative real time RT-PCR and in situ hybridization revealed that STAT1 was expressed in almost all examined organs and tissues, with high expression in gill, spleen, kidney, and heart. The accumulation of STAT1 mRNA in different developmental stages, as determined by real time RT-PCR, increased with development. Conclusion Recent cloning of various cytokine genes and the STAT1 gene of olive flounder here suggest that fish also use the highly specialized JAK-STAT pathway for cytokine signaling. Identification of other STAT genes will elucidate in detail the signal transduction system in this fish. PMID:18578892

  12. Molecular cloning and characterization of an S-adenosylmethionine synthetase gene from Chorispora bungeana.

    PubMed

    Ding, Chenchen; Chen, Tao; Yang, Yu; Liu, Sha; Yan, Kan; Yue, Xiule; Zhang, Hua; Xiang, Yun; An, Lizhe; Chen, Shuyan

    2015-11-10

    S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C. bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C. bungeana S-adenosylmethionine synthetase), from C. bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK. Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C. bungeana.

  13. Molecular cloning and characterization of a Mlo gene in rubber tree (Hevea brasiliensis).

    PubMed

    Qin, Bi; Zheng, Fucong; Zhang, Yu

    2015-03-01

    Mlo gene encodes a plant-specific seven-transmembrane domain protein involved in a variety of cellular processes. In this study, a novel Mlo gene from rubber tree (Hevea brasiliensis), designated HbMlo1, was cloned by RT-PCR in rubber tree. The ORF of HbMlo1 was 1551bp in length, encoding a putative protein of 516 amino acids. HbMlo1 was a typical Mlo protein with seven-transmembrane domain. Sequence comparison between HbMlo1 and other Mlo proteins demonstrated that HbMlo1 shared the highest similarity with the Cucumis melo CmMlo1 and Arabidopsis thaliana AtMlo1 with 75.1% and 71.3% sequence identity, respectively. Phylogenetic analysis revealed that HbMlo1, CmMlo1, AtMlo1, AtMlo13, and AtMlo15 formed into the phylogenetic clade II with 100% bootstrap support value. HbMlo1 transcript exhibited tissue specificity, and it was preferentially expressed in leaf. Furthermore, the amount of HbMlo1 transcript was significantly induced by various phytohormones (including ethephon, methyl jasmonate, salicylic acid, abscisic acid, indole-3-acetic acid, and gibberellic acid), H2O2, and wounding treatments. Under drought stress, HbMlo1 exhibited a complex pattern of regulation. However, HbMlo1 expression did not significantly change during powdery mildew infection. These results suggested that HbMlo1 might play a role in phytohormone signaling and abiotic stress response processes in rubber tree.

  14. Molecular cloning and characterization of a Mlo gene in rubber tree (Hevea brasiliensis).

    PubMed

    Qin, Bi; Zheng, Fucong; Zhang, Yu

    2015-03-01

    Mlo gene encodes a plant-specific seven-transmembrane domain protein involved in a variety of cellular processes. In this study, a novel Mlo gene from rubber tree (Hevea brasiliensis), designated HbMlo1, was cloned by RT-PCR in rubber tree. The ORF of HbMlo1 was 1551bp in length, encoding a putative protein of 516 amino acids. HbMlo1 was a typical Mlo protein with seven-transmembrane domain. Sequence comparison between HbMlo1 and other Mlo proteins demonstrated that HbMlo1 shared the highest similarity with the Cucumis melo CmMlo1 and Arabidopsis thaliana AtMlo1 with 75.1% and 71.3% sequence identity, respectively. Phylogenetic analysis revealed that HbMlo1, CmMlo1, AtMlo1, AtMlo13, and AtMlo15 formed into the phylogenetic clade II with 100% bootstrap support value. HbMlo1 transcript exhibited tissue specificity, and it was preferentially expressed in leaf. Furthermore, the amount of HbMlo1 transcript was significantly induced by various phytohormones (including ethephon, methyl jasmonate, salicylic acid, abscisic acid, indole-3-acetic acid, and gibberellic acid), H2O2, and wounding treatments. Under drought stress, HbMlo1 exhibited a complex pattern of regulation. However, HbMlo1 expression did not significantly change during powdery mildew infection. These results suggested that HbMlo1 might play a role in phytohormone signaling and abiotic stress response processes in rubber tree. PMID:25506769

  15. Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii.

    PubMed

    Lin, Jing-Yun; Ma, Ke-Yi; Bai, Zhi-Yi; Li, Jia-Le

    2013-09-10

    Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a "QPS" and an invariant "WND" motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.

  16. Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase

    SciTech Connect

    Funk, C.D.; Furci, L.; FitzGerald, G.A. )

    1990-08-01

    The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE), is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, the authors have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a M{sub r} 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-12lx) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.

  17. Molecular cloning, functional identification and expressional analyses of FasL in Tilapia, Oreochromis niloticus.

    PubMed

    Ma, Tai-yang; Wu, Jin-ying; Gao, Xiao-ke; Wang, Jing-yuan; Zhan, Xu-liang; Li, Wen-sheng

    2014-10-01

    FasL is the most extensively studied apoptosis ligand. In 2000, tilapia FasL was identified using anti-human FasL monoclonal antibody by Evans's research group. Recently, a tilapia FasL-like protein of smaller molecule weight was predicted in Genbank (XM_003445156.2). Based on several clues drawn from previous studies, we cast doubt on the authenticity of the formerly identified tilapia FasL. Conversely, using reverse transcription polymerase chain reaction (RT-PCR), the existence of the predicted FasL-like was verified at the mRNA level (The Genbank accession number of the FasL mRNA sequence we cloned is KM008610). Through multiple alignments, this FasL-like protein was found to be highly similar to the FasL of the Japanese flounder. Moreover, we artificially expressed the functional region of the predicted protein and later confirmed its apoptosis-inducing activity using a methyl thiazolyl tetrazolium (MTT) assay, Annexin-V/Propidium iodide (PI) double staining, and DNA fragment detection. Supported by these evidences, we suggest that the predicted protein is the authentic tilapia FasL. To advance this research further, tilapia FasL mRNA and its protein across different tissues were quantified. High expression levels were identified in the tilapia immune system and sites where active cell turnover conservatively occurs. In this regard, FasL may assume an active role in the immune system and cell homeostasis maintenance in tilapia, similar to that shown in other species. In addition, because the distribution pattern of FasL mRNA did not synchronize with that of the protein, post-transcriptional expression regulation is suggested. Such regulation may be dominated by potential adenylate- and uridylate-rich elements (AREs) featuring AUUUA repeats found in the 3' untranslated region (UTR) of tilapia FasL mRNA. PMID:24950416

  18. Molecular cloning and expression analysis of an arginine decarboxylase gene from peach (Prunus persica).

    PubMed

    Liu, Ji Hong; Ban, Yusuke; Wen, Xiao-Peng; Nakajima, Ikuko; Moriguchi, Takaya

    2009-01-15

    Arginine decarboxylase (ADC), one of the enzymes responsible for putrescine (Put) biosynthesis, has been shown to be implicated in stress response. In the current paper attempts were made to clone and characterize a gene encoding ADC from peach (Prunus persica (L.) Batsch, 'Akatsuki'). Rapid amplification of cDNA ends (RACE) gave rise to a full-length ADC cDNA (PpADC) with a complete open reading frame of 2178 bp, encoding a 725 amino acid polypeptide. Homology search and sequence multi-alignment demonstrated that the deduced PpADC protein sequence shared a high identity with ADCs from other plants, including several highly conservative motifs and amino acids. Southern blotting indicated that PpADC existed in peach genome as a single gene. Expression levels of PpADC in different tissues of peach (P. persica 'Akatsuki') were spatially and developmentally regulated. Treatment of peach shoots from 'Mochizuki' with exogenous 5 mM Put, an indirect product of ADC, remarkably induced accumulation of PpADC mRNA. Transcripts of PpADC in peach leaves from 'Mochizuki' were quickly induced, either transiently or continuously, in response to dehydration, high salinity (200 mM NaCl), low temperature (4 degrees C) and heavy metal (150 microM CdCl(2)), but repressed by high temperature 37 degrees C) during a 2-day treatment, which changed in an opposite direction when the stresses were otherwise removed with the exception of CdCl(2) treatment. In addition, steady-state of PpADC mRNA could be also transiently up-regulated by abscisic acid (ABA) in 'Mochizuki' leaves. All of these, taken together, suggest that PpADC is a stress-responsive gene and can be considered as a potential target that is genetically manipulated so as to create novel germplasms with enhanced stress tolerance in the future.

  19. Molecular cloning and characterization of an S-adenosylmethionine synthetase gene from Chorispora bungeana.

    PubMed

    Ding, Chenchen; Chen, Tao; Yang, Yu; Liu, Sha; Yan, Kan; Yue, Xiule; Zhang, Hua; Xiang, Yun; An, Lizhe; Chen, Shuyan

    2015-11-10

    S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C. bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C. bungeana S-adenosylmethionine synthetase), from C. bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK. Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C. bungeana. PMID:26205258

  20. Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress)

    PubMed Central

    2004-01-01

    NADH kinase (NADHK; ATP:NADH 2′-phosphotransferase; EC 2.7.1.86), an enzyme that preferentially utilizes NADH as the diphosphonicotinamide nucleotide donor, has been identified for the first time in plants. Low activity (0.4 nmol of NADPH produced/min per mg of protein) was observed in clarified protein extracts from Arabidopsis thaliana (thale cress) cell suspension cultures. However, unlike an NADHK from yeast (Saccharomyces cerevisiae) (POS5), the enzyme from Arabidopsis did not associate with the mitochondria. NADHK was cloned (gi:30699338) from Arabidopsis and studied as a recombinant protein following affinity purification from Escherichia coli. The enzyme had a pH optimum for activity of 7.9 and a subunit molecular mass of 35 kDa. Analytical gel filtration demonstrated that the recombinant enzyme exists as a dimer. Hyperbolic saturation kinetics were observed for the binding of NADH, ATP, free Mg2+ and NAD+, with respective Km values of 0.042, 0.062, 1.16, and 2.39 mM. While NADHK could phosphorylate NADH or NAD+, the specificity constant (Vmax/Km) for NADH was 100-fold greater than for NAD+. The enzyme could utilize UTP, GTP and CTP as alternative nucleotides, although ATP was the preferred substrate. PPi or poly-Pi could not substitute as phospho donors. PPi acted as a mixed inhibitor with respect to both NADH and ATP. NADHK was inactivated by thiol-modifying reagents, with inactivation being decreased in the presence of NADH or ATP, but not NAD+. This study suggests that, in Arabidopsis, NADP+/NADPH biosynthetic capacity could, under some circumstances, become uncoupled from the redox status of the diphosphonicotinamide nucleotide pool. PMID:15347288

  1. Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus.

    PubMed

    Lee, Shyh-Jye; Ju, Chi-Ching; Chu, Shian-Ling; Chien, Ming-Shan; Chan, Tun-Hao; Liao, Wen-Liang

    2007-01-01

    The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven alpha helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia. PMID:17094115

  2. Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L.

    PubMed

    Li, Fupeng; Wu, Baoduo; Qin, Xiaowei; Yan, Lin; Hao, Chaoyun; Tan, Lehe; Lai, Jianxiong

    2014-08-10

    In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype 'TAS-R8'. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production.

  3. Mole ghrelin: cDNA cloning, gene expression, and diverse molecular forms in Mogera imaizumii.

    PubMed

    Satou, Motoyasu; Kaiya, Hiroyuki; Nishi, Yoshihiro; Shinohara, Akio; Kawada, Shin-Ichiro; Miyazato, Mikiya; Kangawa, Kenji; Sugimoto, Hiroyuki

    2016-06-01

    Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1-27], [1-19], [1-16] and [1-15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5'-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals. PMID:27102942

  4. Molecular cloning of a novel multidomain Kunitz-type proteinase inhibitor from the hookworm Ancylostoma caninum.

    PubMed

    Hawdon, John M; Datu, Bennett; Crowell, Melissa

    2003-04-01

    Degenerate oligonucleotide primers derived from conserved serine protease inhibitors were used to amplify a 90-base pair (bp) amplicon from an Ancylostoma caninum adult-stage complementary deoxyribonucleic acid (cDNA) library by polymerase chain reaction (PCR). The amplicon was labeled and used as a probe to screen the library, and a 2,300-bp cDNA clone was identified. The 5' end of the molecule was obtained from adult cDNA by 5'-RACE. The complete sequence named A. caninum Kunitz-type protease inhibitor (Ac-kpi-1) was 2,371 bp and encoded a 759-amino acid open reading frame. The deduced amino acid sequence had a calculated molecular weight of 84,886 Da and contained an amino terminal signal peptide, suggesting that the protein is secreted. Analysis of the predicted protein sequence indicates 12 highly conserved Kunitz-type serine protease inhibitor domains connected by short, conserved spacers. On the basis of sequence analysis, the first 11 domains are predicted to be active serine protease inhibitors based on the P1 amino acid. Domains 5-8 have identical amino acid sequences, and the remaining domains are 38-88% identical. Domain 12 lacks several of the conserved cysteine residues and has an atypical amino acid in the P1 position, suggesting that it is nonfunctional. Reverse transcriptase-PCR indicated that the Ac-kpi-1 messenger ribonucleic acid is present in egg, L1, L3, and adult stages but is most abundant in the adult stage. Ac-KPI-1 is most similar in domain architecture to several extracellular matrix proteins involved in cellular remodeling during insect development. In addition, there are 44 nematode proteins containing one or more Kunitz domains in GenBank, including several with multiple domains.

  5. [Molecular cloning and characteristics of cDNA encoding pig beta6 subunit for FMDV receptor].

    PubMed

    Gao, Shan-Dian; Du, Jun-Zheng; Chang, Hui-Yun; Cong, Guo-Zheng; Shao, Jun-Jun; Shan, Yi Hua; Zhou, Jian-Hua; Xie, Qing-Ge

    2007-09-01

    In order to study the roles of integrin beta6 in Foot-and-Mouth Disease Virus infection, pig integrin beta6 was firstly molecularly cloned from RNA of the tongue and lung of recovered pig infected experimentally with foot-and-mouth-disease virus (FMDV), and was compared with the beta6 gene of other animals available in GenBank at nucleotide and amino acid leves. GeneBank association number of the beta6 gene is EF432729. Pig integrin beta6 gene (2367bp) encodes a polypeptide of 788 amino acids consisting of 9 potential N-linked glycosylation sites, 3 Glycosaminoglycan attachment sites, a cGMP-dependent protein kinase phosphorylation site, 10 Protein kinase C phosphorylation sites, 2 EGF-like domains and 2 cysteine-rich regions. Pig integrin beta6 subunit has a 26-residue putative signal peptide, a 681-residue ectodomain, a 29-residue transmembrane domain, and a 52-residue cytoplasmic domain. 11 mutant nucleotides were found in beta6 gene coding region and 9 amino acids were changed. The nucleotide sequence similarity of integrin beta6 gene between rheses monkey, mouse, Norway rat, dog, guinea pig, human, bovine, sheep is 79.5%, 84.9%, 85.4%, 85.2%, 88.7%, 90.1%, 91.9% and 91.9%, and the amino acid sequence similarity is 93.5%, 88.2%, 88.5%, 88.3%, 91.0%, 92.8%, 93.3% and 93.4% respectively. This study will lay a foundation for understanding the interactions of FMDV with receptors. PMID:18064756

  6. Molecular cloning and identification of the laspartomycin biosynthetic gene cluster from Streptomyces viridochromogenes

    PubMed Central

    Wang, Yang; Chen, Ying; Shen, Qirong; Yin, Xihou

    2011-01-01

    The biosynthetic gene cluster for laspartomycins, a family of 11 amino acid peptide antibiotics, has been cloned and sequenced from Streptomyces viridochromogenes ATCC 29814. Annotation of a segment of 88912 bp of S. viridochromogenes genomic sequence revealed the putative las cluster and its flanking regions which harbor 43 open reading frames. The lpm cluster, which spans approximately 60 kb, consists of 21 open reading frames. Those include four NRPS genes (lpmA/orf18, lpmB/orf25, lpmC/orf26 and lpmD/orf27), four genes (orfs 21, 22, 24 and 29) involved in the lipid tail biosynthesis and attachment, four regulatory genes (orfs 13, 19, 32 and 33) and three putative exporters or self-resistance genes (orfs 14, 20 and 30). In addition, the gene involved in the biosynthesis of the nonproteinogenic amino acid Pip was also identified in the lpm cluster while the genes necessary for the biosynthesis of the rare residue diaminopropionic acid (Dap) were found to reside elsewhere on the chromosome. Interestingly, the dabA, dabB and dabC genes predicted to code for the biosynthesis of the unusual amino acid diaminobutyric acid (Dab) are organized into the lpm cluster even though the Dab residue was not found in the laspartomycins. Disruption of the NRPS lpmC gene completely abolished laspartomycin production in the corresponding mutant strain. These findings will allow molecular engineering and combinatorial biosynthesis approaches to expand the structural diversity of the amphomycin-group peptide antibiotics including the laspartomycins and friulimicins. PMID:21640802

  7. Cloning of porcine GPIHBP1 gene and its tissue expression pattern and genetic effect on adipose traits.

    PubMed

    Xu, Huaming; Tao, Xuelian; Wei, Yingying; Chen, Jianning; Xing, Shuhua; Cen, Wangmin; Wen, Anxiang; Zhu, Li; Tang, Guoqing; Li, Mingzhou; Jiang, Anan; Jiang, Yanzhi; Li, Xuewei

    2015-02-25

    Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism and is transported by glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) from the interstitial spaces to the capillary lumen. Here, we cloned a cDNA and the genomic locus of the porcine GPIHBP1 gene, and investigated its tissue expression pattern and its genetic effects on adipose traits. Porcine GPIHBP1 exhibits a four-exon/three-intron structure, including a 543bp open reading frame that encodes 180 amino acids. The porcine GPIHBP1 protein shows 49%-65% homology and shares the major conserved structural domains of GPIHBP1 proteins in other mammals. Porcine GPIHBP1 mRNA levels were high in the adipose tissue, muscle and lung, and higher mRNA levels were observed in sows compared to boars in adipose tissues of the inner and outer layers of subcutaneous fat, abdominal fat, and suet fat. The mRNA expression pattern of porcine GPIHBP1 and LPL genes was similar in most tissues except for the lung. Thirty six single nucleotide polymorphisms (SNPs) were found in the porcine GPIHBP1 gene. Association analyses showed that the g.-255G>C and g.-626T>G SNPs are associated with intramuscular fat content, and that the g.-1557T>C and g.-1948G>A SNPs are associated with back fat thickness. In conclusion, porcine GPIHBP1 mRNA is abundantly expressed in the adipose tissue, muscle and lung, and gender affects GPIHBP1 mRNA expression levels; furthermore, four GPIHBP1 SNPs are genetic factors affecting adipose traits.

  8. Molecular cloning and expression of novel sulphotransferase-like cDNAs from human and rat brain.

    PubMed Central

    Falany, C N; Xie, X; Wang, J; Ferrer, J; Falany, J L

    2000-01-01

    The sulphotransferase (SULT) gene family is involved with the conjugation of many small drugs, xenobiotics and endogenous compounds. In this report, we describe the cloning and expression of novel cDNAs from human and rat brain, which are structurally related to the SULTs. These cDNAs have been termed 'brain sulphotransferase-like' (BR-STL), because of their similarity to the SULTs and their selective expression in brain tissue. The proteins encoded by the human and rat BR-STL cDNAs (hBR-STL-1 and rBR-STL cDNA respectively), denoted as hBR-STL and rBR-STL, are 98% identical and 99% similar in sequence. The hBR-STL-1 cDNA contains an 852-nt open reading frame encoding a 284-amino-acid protein with a calculated molecular mass of 33083 Da. Northern-blot analyses of RNA isolated from eight human tissues indicate that the hBR-STL message is selectively expressed in brain. Characterization of different brain regions showed that message levels were highest in cortical brain regions. rBR-STL message levels were relatively low in brains of 1-day-old male and female rats, but increased to adult levels in RNA from 7-day-old rats, and remained at that level in adult animals. The hBR-STL and rBR-STL cDNAs were expressed in both Escherichia coli and Sf9 insect cells in the presence or absence of an N-terminal histidine-affinity tag (His-tag). Polyclonal antibodies were raised in chickens against purified His-tagged hBR-STL, and were used to detect the presence of rBR-STL in adult male and female rat brain cytosol. The high degree of sequence conservation, and the selective localization of the BR-STL message in brain, suggest an important function in the central nervous system. PMID:10698717

  9. Molecular cloning and expression analysis of small ubiquitin-like modifier (SUMO) genes from grouper (Epinephelus coioides).

    PubMed

    Xu, Meng; Wei, Jingguang; Chen, Xiuli; Gao, Pin; Zhou, Yongcan; Qin, Qiwei

    2016-01-01

    Small ubiquitin-like modifier (SUMO) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. In the present study, two SUMO homolog genes (EcSUMO1 and EcSUMO2) from grouper (Epinephelus coioides) were cloned and characterized. The full-length sequence of EcSUMO1 was 749 bp in length and contained a predicted open reading frame of 306 bp encoding 101 amino acids with a molecular mass of 11.34 kDa. The full-length sequence of EcSUMO2 was 822 bp in length and contained a predicted open reading frame of 291 bp encoding 96 amino acids with a molecular mass of 10.88 kDa EcSUMO1 shares 44.55% identity with EcSUMO2. EcSUMO1 shares 99%, 90%, and 88% identity with those from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. EcSUMO2 shares 98%, 93%, and 96% identity with those from Anoplopoma fimbria, D.rerio, and H. sapiens, respectively. Quantitative real-time PCR analysis indicated that EcSUMO1 and EcSUMO2 were constitutively expressed in all of the analyzed tissues in healthy grouper, but the expression of EcSUMO2 was higher than that of EcSUMO1. EcSUMO1 and EcSUMO2 were identified as a remarkably (P < 0.01) up-regulated responding to poly(I:C) and Singapore grouper iridovirus (SGIV) stimulation in head kidney of groupers. EcSUMO1 and EcSUMO2 were distributed in both cytoplasm and nucleus in GS cells. Over-expressed EcSUMO1 and EcSUMO2 enhanced SGIV and Red-spotted grouper nervous necrosis virus (RGNNV) replication during viral infection in vitro. Our study was an important attempt to understand the SUMO pathway in fish, which may provide insights into the regulatory mechanism of viral infection in E.coioides under farmed conditions. PMID:26616235

  10. Molecular cloning and expression analysis of small ubiquitin-like modifier (SUMO) genes from grouper (Epinephelus coioides).

    PubMed

    Xu, Meng; Wei, Jingguang; Chen, Xiuli; Gao, Pin; Zhou, Yongcan; Qin, Qiwei

    2016-01-01

    Small ubiquitin-like modifier (SUMO) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. In the present study, two SUMO homolog genes (EcSUMO1 and EcSUMO2) from grouper (Epinephelus coioides) were cloned and characterized. The full-length sequence of EcSUMO1 was 749 bp in length and contained a predicted open reading frame of 306 bp encoding 101 amino acids with a molecular mass of 11.34 kDa. The full-length sequence of EcSUMO2 was 822 bp in length and contained a predicted open reading frame of 291 bp encoding 96 amino acids with a molecular mass of 10.88 kDa EcSUMO1 shares 44.55% identity with EcSUMO2. EcSUMO1 shares 99%, 90%, and 88% identity with those from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. EcSUMO2 shares 98%, 93%, and 96% identity with those from Anoplopoma fimbria, D.rerio, and H. sapiens, respectively. Quantitative real-time PCR analysis indicated that EcSUMO1 and EcSUMO2 were constitutively expressed in all of the analyzed tissues in healthy grouper, but the expression of EcSUMO2 was higher than that of EcSUMO1. EcSUMO1 and EcSUMO2 were identified as a remarkably (P < 0.01) up-regulated responding to poly(I:C) and Singapore grouper iridovirus (SGIV) stimulation in head kidney of groupers. EcSUMO1 and EcSUMO2 were distributed in both cytoplasm and nucleus in GS cells. Over-expressed EcSUMO1 and EcSUMO2 enhanced SGIV and Red-spotted grouper nervous necrosis virus (RGNNV) replication during viral infection in vitro. Our study was an important attempt to understand the SUMO pathway in fish, which may provide insights into the regulatory mechanism of viral infection in E.coioides under farmed conditions.

  11. Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones

    PubMed Central

    Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

    2016-01-01

    Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones. PMID:27555864

  12. Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones.

    PubMed

    Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

    2016-01-01

    Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones. PMID:27555864

  13. Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones.

    PubMed

    Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

    2016-01-01

    Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones.

  14. Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus.

    PubMed

    Ewis, Hosam E; Abdelal, Ahmed T; Lu, Chung-Dar

    2004-03-31

    Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones. The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison. The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da. Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli. The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30. The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol). Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively. With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1). Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family. The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries. PMID:15033540

  15. Molecular cloning and characterization of a complement-depleting factor from king cobra, Ophiophagus hannah.

    PubMed

    Zeng, Lin; Sun, Qian-Yun; Jin, Yang; Zhang, Yong; Lee, Wen-Hui; Zhang, Yun

    2012-09-01

    Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (β chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and β chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure

  16. Molecular cloning and characterization of a complement-depleting factor from king cobra, Ophiophagus hannah.

    PubMed

    Zeng, Lin; Sun, Qian-Yun; Jin, Yang; Zhang, Yong; Lee, Wen-Hui; Zhang, Yun

    2012-09-01

    Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (β chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and β chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure

  17. Molecular cloning and characterization of the DNA of a new human papillomavirus (HPV 30) from a laryngeal carcinoma.

    PubMed

    Kahn, T; Schwarz, E; zur Hausen, H

    1986-01-15

    DNA from a human laryngeal carcinoma was molecularly cloned in Lambda L47. The gene library was screened for human papillomavirus (HPV)-related sequences by hybridization analysis with 32P-labelled HPV 16 DNA at conditions of low stringency (Tm -40 degrees C). One of the clones (4-5) with an insert of 7.8 kb showed cross-hybridization with most of the known HPV types (Tm -40 degrees C), and with several of them even under more stringent conditions (Tm -30 degrees C). No signal was detected under high-stringency conditions (Tm -20 degrees C). The co-linear alignment of clone 4-5 with HPV 16 DNA could be demonstrated by hybridization experiments and also by partial DNA sequence analysis. We conclude that clone 4-5 represents a new HPV type tentatively designated HPV 30. HPV 30 DNA was also detected in 2 genital lesions but not in 41 laryngeal carcinomas analyzed so far. Its presence in other tumor DNA is now under investigation. PMID:3000955

  18. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

    2015-09-01

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  19. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    SciTech Connect

    Woon, J. S. K. Murad, A. M. A. Abu Bakar, F. D.

    2015-09-25

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  20. Utility of epirubicin-incorporating micelles tagged with anti-tissue factor antibody clone with no anticoagulant effect.

    PubMed

    Sugaya, Akinori; Hyodo, Ichinosuke; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Takashima, Hiroki; Sato, Ryuta; Tsumura, Ryo; Furuya, Fumiaki; Yasunaga, Masahiro; Harada, Mitsunori; Tanaka, Ryosuke; Matsumura, Yasuhiro

    2016-03-01

    Tissue factor (TF), an initiator of the extrinsic blood coagulation cascade, is overexpressed in different types of cancer. Tissue factor overexpression is also known as a poor prognostic factor in pancreatic cancer. We recently developed anti-TF antibody (clone1849)-conjugated epirubicin-incorporating micelles (NC-6300), and reported that this anti-TF1849-NC-6300 showed enhanced antitumor activity against TF-high expressed human pancreatic cancer cells, when compared with NC-6300 alone. However, clone 1849 antibody inhibited TF-associated blood coagulation activity. We studied another anti-TF antibody, clone 1859, which had no effect on blood coagulation and prepared anti-TF1859-NC-6300. In addition, to determine the optimum size of the antibody fragment to conjugate with NC-6300, three forms of the 1859 antibody (whole IgG, F[ab']2 , and Fab') were conjugated to NC-6300. The antitumor effect of each anti-TF1859-NC-6300 was studied in vitro and in vivo, using two human pancreatic cancer cell lines, BxPC3 with high-expressed TF, and SUIT2 with low levels of TF. In vitro, all forms of anti-TF1859-NC-6300 showed higher cytocidal effects than NC-6300 in BxPC3, whereas this enhanced effect was not observed in SUIT2. Likewise, all forms of anti-TF1859-NC-6300 significantly suppressed tumor growth when compared to NC-6300 in the BxPC3, but not in the SUIT2, xenograft model. Among the three forms of conjugates, anti-TF1859-IgG-NC-6300 had a higher antitumor tendency in TF-high expressed cells. Thus, we have confirmed an enhanced antitumor effect of anti-TF1859-NC-6300 in a TF-high expressing tumor; anti-TF1859-IgG-NC-6300 could be used to simplify the manufacturing process of the antibody-micelle conjugation for future clinical studies.

  1. Molecular cloning, characterization and expression analysis of thrombospondin gene from Penaeus monodon.

    PubMed

    Zhou, FaLin; Zheng, Liming; Zhang, Dianchang; Huang, JianHua; Qiu, Lihua; Yang, QiBin; Jiang, ShiGui

    2011-06-01

    In present study, a thrombospondin gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length P. monodon thrombospondin (PmTSP) cDNA contained a 5' untranslated region (UTR) of 9 bp, an open reading frame (ORF) of 2778 bp encoding a polypeptide of 925 amino acids with molecular mass 100.57 kDa, and a 3'UTR of 99 bp. ScanProsite analysis indicated that PmTSP contained four chitin-binding type-II domains, an EGF-like domain, eight thrombospondin type-III repeats and one thrombospondin C-terminal domain. Homology analysis of the deduced amino acid sequence of the PmTSP with other known TSP sequences by MatGAT software revealed that the PmTSP shows very high homology with the sequences of Fennerpenaeus chinensis (89.9% similarity, 83.8% identity). Analysis of the tissue expression pattern of the PmTSP gene showed that the PmTSP mRNA was expressed in all tested tissues, including hepatopancreas, ovary, muscle, intestine, neurosecretory organ in eyestalk, neurosecretory organ in brain, stomach, and heart, with highest level in the ovary. Furthermore, the PmTSP expression was found to be of high level in six development stages of the ovary. The results indicated that PmTSP might play an important role in ovarian development.

  2. Molecular cloning, computational and expression analysis of anthocyanidin reductase in tea (Camellia sinensis).

    PubMed

    Thirugnanasambantham, Krishnaraj; Muralidaran, Senguttuvan; Mandal, Abul Kalam Azad

    2014-09-01

    Tea [Camellia sinensis (L.) O. Kuntze] is one of the most popular non-alcoholic beverages rich in phenolic compounds, which includes epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and catechin (C). Anthocyanidin reductase (ANR) is responsible for catechin biosynthesis in plants, and analysis of its protein sequences and structures will be valuable for further research in the field. We have screened our dormant bud-specific complementary DNA (cDNA) library and reported 1,322-bp cDNA encoding CsANR. Analysis of the sequence revealed the presence of 1,011-bp open reading frame with coding capacity for a polypeptide of 337 amino acids, flanked by 1,123- and 196-bp 5' and 3' untranslated regions, respectively. Theoretical molecular weight (MW) and isoelectric point (pI) of the deduced ANR protein were predicted (using ProtParam) to be 36.4 kDa and 6.54. For the first time, we have reported 3D model of ANR from C. sinensis. Quality of the predicted model was analysed with PROCHECK analysis. Molecular docking of modelled ANR revealed similar binding pockets for both substrates and products. Expression analyses of CsANR and accumulation pattern of catechins were observed to be varied with developmental age of tissue and seasonal condition. Variation in accumulation pattern of catechins and its fractions was found to be correlated with expression pattern of ANR.

  3. Molecular cloning, characterization and expression of the phenylalanine ammonia-lyase gene from Juglans regia.

    PubMed

    Xu, Feng; Deng, Guang; Cheng, Shuiyuan; Zhang, Weiwei; Huang, Xiaohua; Li, Linling; Cheng, Hua; Rong, Xiaofeng; Li, Jinbao

    2012-01-01

    Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenypropanoid pathway. A full-length cDNA of PAL gene was isolated from Juglans regia for the first time, and designated as JrPAL. The full-length cDNA of the JrPAL gene contained a 1935bp open reading frame encoding a 645-amino-acid protein with a calculated molecular weight of about 70.4 kD and isoelectric point (pI) of 6.7. The deduced JrPAL protein showed high identities with other plant PALs. Molecular modeling of JrPAL showed that the 3D model of JrPAL was similar to that of PAL protein from Petroselinum crispum (PcPAL), implying that JrPAL may have similar functions with PcPAL. Phylogenetic tree analysis revealed that JrPAL shared the same evolutionary ancestor of other PALs and had a closer relationship with other angiosperm species. Transcription analysis revealed that JrPAL was expressed in all tested tissues including roots, stems, and leaves, with the highest transcription level being found in roots. Expression profiling analyses by real-time PCR revealed that JrPAL expression was induced by a variety of abiotic and biotic stresses, including UV-B, wounding, cold, abscisic acid and salicylic acid.

  4. Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes.

    PubMed Central

    Kawamura, M; McVicar, D W; Johnston, J A; Blake, T B; Chen, Y Q; Lal, B K; Lloyd, A R; Kelvin, D J; Staples, J E; Ortaldo, J R

    1994-01-01

    Protein-tyrosine kinases (PTKs) are critical enzymes for receptor-mediated signaling in lymphocytes. Because natural killer (NK) cells are large granular lymphocytes with specialized effector function, we set out to identify PTKs preferentially expressed in these cells. One such PTK was identified and molecularly cloned. The predicted amino acid sequence shows that this kinase lacks SH2 or SH3 domains typical of src family kinases but has tandem nonidentical catalytic domains, indicating that it is a member of the Janus family of PTKs. Immunoprecipitation using antiserum generated against a peptide corresponding to the deduced amino acid sequence of this gene revealed a kinase with a molecular weight of approximately 125,000. The pattern of expression of this kinase contrasted sharply with that of other Janus kinases, which are ubiquitously expressed. The kinase described in the present study was found to be more limited in its expression; expression was found in NK cells and an NK-like cell line but not in resting T cells or in other tissues. In contrast, stimulated and transformed T cells expressed the gene, suggesting a role in lymphoid activation. Because of its homology and tissue expression, we have tentatively termed this PTK gene L-JAK for leukocyte Janus kinase. Images PMID:8022790

  5. Molecular cloning of the breakpoints of a complex Philadelphia chromosome translocation: identification of a repeated region on chromosome 17.

    PubMed Central

    McKeithan, T W; Warshawsky, L; Espinosa, R; LeBeau, M M

    1992-01-01

    Complex translocations in chronic myelogenous leukemia involve various chromosomes, in addition to chromosomes 9 and 22, in a nonrandom fashion. We have analyzed the DNA from leukemia cells characterized by a complex translocation, t(9;22;10;17)(q34;q11;p13;q21), by using the techniques of Southern blot hybridization, in situ hybridization, and molecular cloning; one of the breakpoints is at 17q21, a band that is frequently involved in complex 9;22 translocations. All of the breakpoint junctions and the corresponding normal sequences from the four involved chromosomes have been molecularly cloned. Restriction mapping is consistent with a simple concerted exchange of chromosomal material among the four chromosomes, except that additional changes appeared to have occurred within the chromosome 17 sequences. The cloned sequences on chromosome 17 at band q21 were found to be repeated in normal cells. By fluorescence in situ hybridization, a strong signal is seen at 17q21, but a weaker signal is also present at 17q23. By comparison with other primate species, an inversion in chromosome 17 during evolution appears to be responsible for the splitting of the cluster of repeat units in normal human cells. Images PMID:1594595

  6. Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

    PubMed

    Fu, J J; Mei, Z Q; Tania, M; Yang, L Q; Cheng, J L; Khan, M A

    2015-05-25

    The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

  7. Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis.

    PubMed

    Liu, Fengsong; Li, Fuhua; Dong, Bo; Wang, Xiaomei; Xiang, Jianhai

    2009-03-01

    A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp. PMID:18163220

  8. RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.

    PubMed

    Trujillo-Provencio, Casilda; Powers, TuShun R; Sultemeier, David R; Ramirez-Gordillo, Daniel; Serrano, Elba E

    2016-01-01

    The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs. PMID:27259922

  9. RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.

    PubMed

    Trujillo-Provencio, Casilda; Powers, TuShun R; Sultemeier, David R; Ramirez-Gordillo, Daniel; Serrano, Elba E

    2016-01-01

    The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs.

  10. Molecular Epidemiology of Staphylococcus aureus among Patients with Skin and Soft Tissue Infections in Two Chinese Hospitals

    PubMed Central

    Gu, Fei-Fei; Chen, Ye; Dong, De-Ping; Song, Zhen; Guo, Xiao-Kui; Ni, Yu-Xing; Han, Li-Zhong

    2016-01-01

    Background: Staphylococcus aureus is one of the predominant causes of skin and soft tissue infections (SSTIs), but limited data were available regarding the characterization of S. aureus from SSTIs patients in Jiangsu Province in China. We aimed to investigate the molecular epidemiology of S. aureus among SSTIs patients in two hospitals of Jiangsu Province. Methods: Sixty-two patients with SSTIs from two Chinese hospitals in Jiangsu Province were enrolled in this study, and 62 S. aureus isolates were collected from February 2014 to January 2015. S. aureus isolates were characterized by antimicrobial susceptibility testing, toxin gene detection, and molecular typing with sequence type, Staphylococcus protein A gene type, accessory gene regulator (agr) group, and Staphylococcal cassette chromosome mec type. Results: Sixteen (25.8%) methicillin-resistant S. aureus (MRSA) isolates were detected, and there was no isolate found resistant to vancomycin, teicoplanin, sulfamethoxazole-trimethoprim, and linezolid. The sei was the toxin gene most frequently found, and no lukS/F-PV-positive isolates were detected among the SSTIs’ patients. Molecular analysis revealed that ST398 (10/62, 16.1%; 2 MRSA and 8 methicillin-susceptible S. aureus) to be the dominant clone, followed by ST5 (8/62, 12.9%) and ST7 (8/62, 12.9%). Conclusions: The livestock ST398 was the most common clone among patients with S. aureus SSTIs in Jiangsu Province, China. Surveillance and further studies on the important livestock ST398 clone in human infections are necessarily requested. PMID:27647191

  11. Cloning and tissue distribution of appetite-regulating peptides in pirapitinga (Piaractus brachypomus).

    PubMed

    Volkoff, H

    2015-10-01

    Pirapitinga (or red-bellied pacu, Piaractus brachypomus, Characiforme, Serrasalmidae) is an economically important South American fish for which the endocrine mechanism of the regulation of feeding has never been examined. To better understand these mechanisms, cDNAs encoding the appetite-regulating peptides orexin, cocaine- and amphetamine-regulated transcript (CART), apelin, cholecystokinin (CCK), peptide YY (PYY), leptin and ghrelin were isolated in pirapitinga and their mRNA distributions examined in peripheral tissues and brain. When compared to other fish, the sequences obtained for all peptides were most similar to those of other Characiforme fish (i.e. Mexican cavefish) and Siluriformes (catfish) as well as Cypriniformes (i.e. goldfish, zebrafish). All peptides were widely expressed within the brain. With the exception of CART, which was only expressed in brain, the mRNAs of all peptides were present in several peripheral tissues, including gastrointestinal tract, kidneys and gills. The widespread and peptide-specific distributions suggest that each peptide might have distinct physiological actions in the brain and on peripheral tissues, in particular on the gastrointestinal tract, which include feeding regulation. This preliminary study opens new avenues for further functional studies on the endocrine regulation of feeding in Serrasalmidae fish, including pirapitinga. PMID:25846408

  12. Molecular cloning, expression, and signaling pathway of four melanin-concentrating hormone receptors from Xenopus tropicalis.

    PubMed

    Kobayashi, Yuki; Hamamoto, Akie; Hirayama, Tomo; Saito, Yumiko

    2015-02-01

    Melanin-concentrating hormone (MCH) mainly regulates feeding in mammals and pigmentation in teleosts. It acts via two G-protein-coupled receptors, MCH receptor 1 (MCHR1) and MCHR2. Although many studies exploring the MCH system in teleosts and mammals have been carried out, studies on other organisms are limited. In this study, we cloned and characterized four MCHR subtypes from the diploid species Xenopus tropicalis (X-MCHRs; X-MCHR1a, R1b, R2a, and R2b). According to a phylogenetic tree of the X-MCHRs, X-MCHR1a and R2a are close to mammalian MCHRs, while X-MCHR1b and R2b are close to teleostean MCHRs. We previously reported that the G-protein coupling capacity of the MCHR subtypes differed between mammals (R1: Gαi/o and Gαq; R2: Gαq) and teleosts (R1: Gαq; R2: Gαi/o and Gαq) in mammalian cell-based assays. By using Ca(2+) mobilization assays with pertussis toxin in CHO dhfr(-) cells, we found that X-MCHR1a promiscuously coupled to both Gαi/o and Gαq, while X-MCHR1b and R2a exclusively coupled to Gαq. However, no Ca(2+) influx was detected in cells transfected with X-MCHR2b. Reverse transcription-PCR showed that the X-MCHR mRNAs were expressed in various tissues. In particular, both X-MCHR1b and R2b were exclusively found in melanophores of the dorsal skin. In skin pigment migration assays, melanophores were weakly aggregated at low concentrations but dispersed at high concentrations of MCH, suggesting possible interactions between X-MCHR1b and R2b for the regulation of body color. These findings demonstrate that X. tropicalis has four characteristic MCHRs and will be useful for elucidating the nature of MCHR evolution among vertebrates.

  13. GENE EXPRESSION PROFILING OF ACCESSIBLE SURROGATE TISSUES TO MONITOR MOLECULAR CHANGES IN INACCESSIBLE TARGET TISSUES FOLLOWING TOXICANT EXPOSURE

    EPA Science Inventory

    Gene Expression Profiling Of Accessible Surrogate Tissues To Monitor Molecular Changes In Inaccessible Target Tissues Following Toxicant Exposure
    John C. Rockett, Chad R. Blystone, Amber K. Goetz, Rachel N. Murrell, Judith E. Schmid and David J. Dix
    Reproductive Toxicology ...

  14. Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

    PubMed

    Wiśniewski, Marcin; Lapiński, Maciej; Zdziarska, Anna; Długosz, Ewa; Bąska, Piotr

    2014-08-01

    Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor. PMID

  15. SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

    PubMed Central

    Stewart, Andrew K.; Shmukler, Boris E.; Vandorpe, David H.; Reimold, Fabian; Heneghan, John F.; Nakakuki, M.; Akhavein, Arash; Ko, Shigeru; Ishiguro, Hiroshi

    2011-01-01

    The secretin-stimulated human pancreatic duct secretes HCO3−-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO3− secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl−/HCO3− exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO3− or more, mouse and rat ducts secrete ∼40–70 mM HCO3−. Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO3− secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl−/Cl− exchange and electroneutral Cl−/HCO3− exchange. gpSlc26a6 in Xenopus oocytes mediated Cl−/Cl− exchange and bidirectional exchange of Cl− for oxalate and sulfate, but Cl−/HCO3− exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl−, oxalate, and sulfate transport but no detectable Cl−/HCO3− exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of 36Cl− influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO3− secretion in species that share a high HCO3− secretory output. PMID:21593449

  16. Molecular cloning and characterization of cathepsin L from freshwater mussel, Cristaria plicata.

    PubMed

    Hu, Xiaojuan; Hu, Xiangping; Hu, Baoqing; Wen, Chungen; Xie, Yanhai; Wu, Dan; Tao, Zhiying; Li, Aihua; Gao, Qian

    2014-10-01

    Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. The Cathepsin L cDNA and genome of Cristaria plicata(CpCL) was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends (RACE) PCR. The genomic DNA was 9353 bp long and had a total of six introns and seven exons. The full-length cDNA of CpCL was 1144 bp, the cDNA contained a 5' untranslated region (UTR) of 34 nucleotides, the 3' UTR of 108 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1002 bp, encoding 333 amino acid residues with 37.65 kDa predicted molecular weight. The theoretical isoelectric point was 8.61. The prepro-cathepsin L was consisted of a typical signal peptide (Met1-Gly20), a pro-region peptide (Leu21-Glu116) and a mature peptide (Tyr117-Val333). Many members of the papain family possessed of a proline residue at position 2 in the mature enzymem, this was also observed in CpCL. The preproprotein included an oxyanion hole (Gln 135), the active center formed by Cys141, His280 and Asn 300, the potential N-glycosylation site (Asn38, Asn 113 and Asn 272) and the conserved GCXGG motifs, which was characteristic of cathepsin, the conserved ERWNIN and GNFD motifs, which were characteristic for cathepsin L. Homology analysis revealed that the CpCL shared 49-87% identity to other known cathepsin L sequences. The phylogenetic tree showed that the CpCL clustered with the invertebrate cathepsin L cysteine proteases, and was closely related to the cathepsin L of Hyriopsis cumingii. The expression of CpCL mRNA was detected in hepatopancreas, hemocytes, mantle, gills and adductor muscle, and the higher expression level was in hepatopancreas. After A. hydrophila stimulation, the expression of the CpCL mRNA was up-regulated in hemocytes and hepatopancreas, and the expression level was significantly lower in gill than one after PBS challenge group. PMID:25038281

  17. Molecular cloning and characterization of potato spindle tuber viroid cDNA sequences

    PubMed Central

    Owens, Robert A.; Cress, Dean E.

    1980-01-01

    Double-stranded cDNA has been synthesized from a polyadenylylated potato spindle tuber viroid (PSTV) template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC)·oligo(dG)-tailing procedure. Tetracycline-resistant ampicillin-sensitive transformants contained sequences complementary to PSTV [32P]cDNA, and one recombinant clone (pDC-29) contains a 460-base-pair insert. This cloned double-stranded PSTV cDNA contains the cleavage sites for six restriction endonucleases predicted by the published primary sequence of PSTV as well as one additional site each for Ava I, Hae III, Hpa II, and Sma I. The additional Ava I, Hpa II, and Sma I sites are explained by the presence of a second C-C-C-G-G-G sequence in the cloned double-stranded cDNA. The largest fragment released by Hae III digestion contains approximately 360 base pairs. These results suggest that we have cloned almost the entire sequence of PSTV, but the sequence cloned differs slightly from that published. Hybridization probes derived from pDC-29 insert have allowed detection and preliminary characterization of RNA molecules having the same size as PSTV but the opposite polarity. This RNA is present during PSTV replication in infected tomato cells. Images PMID:16592877

  18. Molecular characterization, tissue distribution, and immune reaction expression of karyopherins in the domestic silkworm (Bombyx mori).

    PubMed

    Li, J; Wang, L; Qian, C; Zhang, C F; Dai, L S; Liu, Q N; Wei, G Q; Sun, Y; Liu, D R; Zhu, B J; Liu, C L

    2015-01-01

    Karyopherins, including alpha and beta types, are transport proteins in the eukaryotic cell that carry cargoes across nuclear pore complexes into or out of the nucleus. In this study, full open reading frames of one beta and three alpha types of karyopherin were cloned from cDNA of the domestic silkworm (Bombyx mori). The one beta and three alpha types' open reading frames were 2661, 1563, 1515, and 1551 base pairs long, respectively, and coded 886, 520, 504, and 516 amino acids, respectively. The alphas all had one importin-beta-binding (IBB) domain, and eight, four, or seven armadillo/beta-catenin-like repeats. The beta had 19 HEAT repeat domains, which constructed one importin-beta-N-terminal domain and one IBB domain. The recombinant proteins were expressed in Escherichia coli cells. The molecular weight of the beta type was approximately 100 kDa, and the alphas weighed approximately 60 kDa. Phylogenic tree construction revealed that the alphas could be classified into three known karyopherin-alpha subfamilies. We detected mRNA of the four karyopherins in normal 3rd day of 5th instar larvae, and in larvae injected with Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi using real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction, and found that the four karyopherins were widely distributed, but their expression levels were related to tissues type, the microbe injected, and the time point.

  19. Molecular characterization, tissue distribution, and immune reaction expression of karyopherins in the domestic silkworm (Bombyx mori).

    PubMed

    Li, J; Wang, L; Qian, C; Zhang, C F; Dai, L S; Liu, Q N; Wei, G Q; Sun, Y; Liu, D R; Zhu, B J; Liu, C L

    2015-01-01

    Karyopherins, including alpha and beta types, are transport proteins in the eukaryotic cell that carry cargoes across nuclear pore complexes into or out of the nucleus. In this study, full open reading frames of one beta and three alpha types of karyopherin were cloned from cDNA of the domestic silkworm (Bombyx mori). The one beta and three alpha types' open reading frames were 2661, 1563, 1515, and 1551 base pairs long, respectively, and coded 886, 520, 504, and 516 amino acids, respectively. The alphas all had one importin-beta-binding (IBB) domain, and eight, four, or seven armadillo/beta-catenin-like repeats. The beta had 19 HEAT repeat domains, which constructed one importin-beta-N-terminal domain and one IBB domain. The recombinant proteins were expressed in Escherichia coli cells. The molecular weight of the beta type was approximately 100 kDa, and the alphas weighed approximately 60 kDa. Phylogenic tree construction revealed that the alphas could be classified into three known karyopherin-alpha subfamilies. We detected mRNA of the four karyopherins in normal 3rd day of 5th instar larvae, and in larvae injected with Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi using real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction, and found that the four karyopherins were widely distributed, but their expression levels were related to tissues type, the microbe injected, and the time point. PMID:26535618

  20. Molecular cloning and characterization of a novel Y-box gene from Sepiella maindroni.

    PubMed

    Si, H P; Wang, C L; Zhang, Y Y; Mu, C K; Zhan, P P; Li, R H; Song, W W

    2015-01-01

    Y-box proteins are a family of highly conserved nucleic acid binding proteins that interact with genome and transcription product to modulate the transcriptional and translational processes. In the present study, a complete mRNA of Y-box binding protein (designated SmYB) was obtained from Sepiella maindroni by amplification of flanking sequences. The full size of SmYB cDNA was 1502 bp, including 99 bp at the 5ꞌ untranslated region (UTR), a 3ꞌ UTR of 821 bp with a poly (A) tail, and an open reading frame of 582 bp, encoding a polypeptide of 193 amino acids with the predicted molecular weight of 16.48 kDa. The conserved cold-shock domain and two known RNA binding motifs identified in SmYB strongly suggested that SmYB was a new member of Y-box proteins. Quantitative real-time PCR was performed to examine the expression of SmYB mRNA in various tissues, embryos, and its temporal expression in liver after cold shock. The mRNA transcript of SmYB was detected in all examined tissues, with the highest expression level in testis and ovary. SmYB was abundant in early developmental stages of S. maindroni embryos but diminished in the late post-embryonic development. In addition, cold-shock treatment upregulated the transcription of SmYB mRNA in liver. These results demonstrated that SmYB is involved in embryonic development of S. maindroni and its tolerance to acute low temperatures. PMID:26125774

  1. Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules

    PubMed Central

    Girard, Jean-Philippe; Baekkevold, Espen S.; Feliu, Jacques; Brandtzaeg, Per; Amalric, François

    1999-01-01

    High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na+-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na+/sulfate cotransporter, NaSi-1, as well as with the human and rat Na+/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na+-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na+-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV. PMID:10535998

  2. Construction and characterization of a human T-cell lymphotropic virus type 3 infectious molecular clone.

    PubMed

    Chevalier, Sébastien Alain; Ko, Nga Ling; Calattini, Sara; Mallet, Adeline; Prévost, Marie-Christine; Kehn, Kylene; Brady, John N; Kashanchi, Fatah; Gessain, Antoine; Mahieux, Renaud

    2008-07-01

    We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24(gag) protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.

  3. Molecular clock integration of brown adipose tissue formation and function.

    PubMed

    Nam, Deokhwa; Yechoor, Vijay K; Ma, Ke

    2016-01-01

    The circadian clock is an essential time-keeping mechanism that entrains internal physiology to environmental cues. Despite the well-established link between the molecular clock and metabolic homeostasis, an intimate interplay between the clock machinery and the metabolically active brown adipose tissue (BAT) is only emerging. Recently, we came to appreciate that the formation and metabolic functions of BAT, a key organ for body temperature maintenance, are under an orchestrated circadian clock regulation. Two complementary studies from our group uncover that the cell-intrinsic clock machinery exerts concerted control of brown adipogenesis with consequent impacts on adaptive thermogenesis, which adds a previously unappreciated temporal dimension to the regulatory mechanisms governing BAT development and function. The essential clock transcriptional activator, Bmal1, suppresses adipocyte lineage commitment and differentiation, whereas the clock repressor, Rev-erbα, promotes these processes. This newly discovered temporal mechanism in fine-tuning BAT thermogenic capacity may enable energy utilization and body temperature regulation in accordance with external timing signals during development and functional recruitment. Given the important role of BAT in whole-body metabolic homeostasis, pharmacological interventions targeting the BAT-modulatory activities of the clock circuit may offer new avenues for the prevention and treatment of metabolic disorders, particularly those associated with circadian dysregulation. PMID:27385482

  4. Molecular clock integration of brown adipose tissue formation and function

    PubMed Central

    Nam, Deokhwa; Yechoor, Vijay K.; Ma, Ke

    2016-01-01

    Abstract The circadian clock is an essential time-keeping mechanism that entrains internal physiology to environmental cues. Despite the well-established link between the molecular clock and metabolic homeostasis, an intimate interplay between the clock machinery and the metabolically active brown adipose tissue (BAT) is only emerging. Recently, we came to appreciate that the formation and metabolic functions of BAT, a key organ for body temperature maintenance, are under an orchestrated circadian clock regulation. Two complementary studies from our group uncover that the cell-intrinsic clock machinery exerts concerted control of brown adipogenesis with consequent impacts on adaptive thermogenesis, which adds a previously unappreciated temporal dimension to the regulatory mechanisms governing BAT development and function. The essential clock transcriptional activator, Bmal1, suppresses adipocyte lineage commitment and differentiation, whereas the clock repressor, Rev-erbα, promotes these processes. This newly discovered temporal mechanism in fine-tuning BAT thermogenic capacity may enable energy utilization and body temperature regulation in accordance with external timing signals during development and functional recruitment. Given the important role of BAT in whole-body metabolic homeostasis, pharmacological interventions targeting the BAT-modulatory activities of the clock circuit may offer new avenues for the prevention and treatment of metabolic disorders, particularly those associated with circadian dysregulation. PMID:27385482

  5. Molecular clock integration of brown adipose tissue formation and function.

    PubMed

    Nam, Deokhwa; Yechoor, Vijay K; Ma, Ke

    2016-01-01

    The circadian clock is an essential time-keeping mechanism that entrains internal physiology to environmental cues. Despite the well-established link between the molecular clock and metabolic homeostasis, an intimate interplay between the clock machinery and the metabolically active brown adipose tissue (BAT) is only emerging. Recently, we came to appreciate that the formation and metabolic functions of BAT, a key organ for body temperature maintenance, are under an orchestrated circadian clock regulation. Two complementary studies from our group uncover that the cell-intrinsic clock machinery exerts concerted control of brown adipogenesis with consequent impacts on adaptive thermogenesis, which adds a previously unappreciated temporal dimension to the regulatory mechanisms governing BAT development and function. The essential clock transcriptional activator, Bmal1, suppresses adipocyte lineage commitment and differentiation, whereas the clock repressor, Rev-erbα, promotes these processes. This newly discovered temporal mechanism in fine-tuning BAT thermogenic capacity may enable energy utilization and body temperature regulation in accordance with external timing signals during development and functional recruitment. Given the important role of BAT in whole-body metabolic homeostasis, pharmacological interventions targeting the BAT-modulatory activities of the clock circuit may offer new avenues for the prevention and treatment of metabolic disorders, particularly those associated with circadian dysregulation.

  6. Molecular cloning, mRNA expression, and characterization of heat shock protein 10 gene from humphead snapper Lutjanus sanguineus.

    PubMed

    Zhang, Xinzhong; Dai, Liping; Wu, Zaohe; Jian, Jichang; Lu, Yishan

    2011-09-01

    Heat shock protein 10 (HSP10) gene of humphead snapper (Lutjanus sanguineus), designated as ByHSP10, was cloned by rapid amplification of cDNA ends (RACE) techniques with the primers designed from the known EST sequence identified from the subtracted cDNA library of the head kidney of humphead snapper. Sequence analysis showed the full length cDNA of ByHSP10 was 529bp, containing a 5' terminal untranslated region (UTR) of 51bp, a 3' terminal UTR of 181bp, and an open reading frame (ORF) of 297bp encoding a polypeptide of 99 amino acids. Based on the deduced amino acid sequence, the theoretical molecular mass of ByHSP10 was calculated to be 10.92kDa with an isoelectric point of 9.46. Moreover, chaperonins hsp10/cpn10 signature was found in the amino acids sequence of ByHSP10 by PredictProtein. BLAST analysis revealed that the amino acids of ByHSP10 had the highest homology of 88% compared with other HSP10s. Fluorescent real-time quantitative RT-PCR was used to examine the expression of ByHSP10 gene in eight kinds of tissues of humphead snapper after the challenge with Vibrio harveyi. There was a clear time-dependent expression pattern of ByHSP10 in head kidney, spleen and thymus after bacteria challenge. The expression of mRNA reached the maximum level at the time point of 9h, 6h and 24h, respectively and then returned to control level in 36h. The up-regulated mRNA expression of ByHSP10 in humphead snapper after bacteria challenge indicated that the HSP10 gene was inducible and might be involved in immune response. A phylogenetic tree was constructed based on the ORF nucleotide sequences of HSP10 for 30 species. The relatonships among them were generally in agreement with the traditional taxonomy which suggested that HSP10 genes could aid in the system classification research.

  7. Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco.

    PubMed

    Ke, F; Zhang, H B; Wang, Y; Hou, L F; Dong, H J; Wang, Z F; Pan, G W; Cao, X Y

    2016-09-01

    This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR). PMID:27418461

  8. Molecular cloning of estrogen receptor alpha (ERalpha; ESR1) of the Japanese giant salamander, Andrias japonicus.

    PubMed

    Katsu, Yoshinao; Kohno, Satomi; Oka, Tomohiro; Mitsui, Naoko; Tooi, Osamu; Santo, Noriaki; Urushitani, Hiroshi; Fukumoto, Yukio; Kuwabara, Kazushi; Ashikaga, Kazuhide; Minami, Shinji; Kato, Shigeaki; Ohta, Yasuhiko; Guillette, Louis J; Iguchi, Taisen

    2006-09-26

    Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander (Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ERalpha (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERalpha mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan.

  9. Molecular cloning and expression analysis of a gene for sucrose transporter from pear (Pyrus bretschneideri Rehd.) fruit.

    PubMed

    Zhang, Huping; Zhang, Shujun; Qin, Gaihua; Wang, Lifen; Wu, Tao; Qi, Kaijie; Zhang, Shaoling

    2013-12-01

    Here we report the cloning of a sucrose transporter cDNA from pear (Pyrus bretschneideri Rehd. cv 'Yali') fruit and an analysis of the expression of the gene. A cDNA clone, designated PbSUT1 was identified as a sucrose transporter cDNA from its sequence homology at the amino acid level to sucrose transporters that have been cloned from other higher plant species. PbSUT1 potentially encoded a protein of 499 amino acid residues with a predicted molecular mass of 53.4 kDa and an isoelectric point (pI) of 9.21. Phylogenetic analysis revealed that the PbSUT1 belonged to type III SUTs and was more closely related to the MdSUT1 from apple fruit. Some major facilitator superfamily (MFS)-specific sequence motifs were found in the predicted PbSUT1 peptides, and an MFS_1 domain was located at the amino acid positions of 29-447 of the sequence. A study of gene expression along fruit development showed that PbSUT1 transcripts are present at all stages but significantly increase before fruit enlargement and during the ripening process with increasing sucrose levels. In contrast, the expression levels don't change much during the period of rapid fruit growth. This work shows that sucrose transporter may play a role in the accumulation of sugars during maturation and in maintaining the internal cellular distribution.

  10. Molecular cloning and functional characterization of the diapause hormone receptor in the corn earworm Helicoverpa zea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diapause hormone (DH) in the heliothine moth has shown its activity in termination of pupal diapause, while the orthology in the silkworm is known to induce embryonic diapause. In the current study, we cloned the diapause hormone receptor from the corn earworm Helicoverpa zea (HzDHr) and tested ...

  11. Molecular Cloning, Expression and Genome Organization of Channel Catfish (Ictalurus punctatus) Matrix Metalloproteinase-9

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the course of studying pathogenesis of enteric septicemia of catfish, we noted that channel catfish matrix metalloproteinase-9 (MMP-9) gene was up-regulated after Edwardsiella ictaluri infection. In this study, we cloned, sequenced using the RACE (rapid amplification of cDNA ends) method and cha...

  12. Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

    Technology Transfer Automated Retrieval System (TEKTRAN)

    : Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

  13. Molecular cloning of Reteplase and its expression in E. coli using tac promoter

    PubMed Central

    Aghaabdollahian, Safieh; Rabbani, Mohammad; Ghaedi, Kamran; Sadeghi, Hamid Mir Mohammad

    2014-01-01

    Background and Aims: This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression. Materials and Methods: Reteplase cDNA was amplified by polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE. Results: Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG. Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli. PMID:25298959

  14. Molecular Cloning and Characterisation of Heparanase mRNA in Porcine Placenta Throughout Gestation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The placenta contains a complex extracellular matrix composed of several glycosaminoglycans including heparan sulfate (HS). Heparanase (HPSE) is an endoglycosidase that specifically degrades HS. The objective of this study was to clone cDNA encoding porcine HPSE and characterize the expression lev...

  15. Molecular cloning and mRNA expression analysis of antizyme inhibitor 1 in the ovarian follicles of the Sichuan white goose.

    PubMed

    Ma, Rong; Jiang, Dongmei; Kang, Bo; Bai, Lin; He, Hui; Chen, Ziyu; Yi, Zhixin

    2015-08-15

    Antizyme inhibitor 1 (Azin1) plays critical roles in various cellular pathways, including ornithine decarboxylase regulation, polyamine anabolism and uptake and cell proliferation. However, the molecular characteristics of the AZIN1 gene and its expression profile in goose tissues and ovarian follicles have not been reported. In this study, the AZIN1 cDNA of the Sichuan white goose (Anser cygnoides) was cloned, and analyzed for its phylogenetic and physiochemical properties. The expression profile of AZIN1 mRNA in geese tissues and ovarian follicles were examined using quantitative real-time PCR. The results showed that the open reading frame of the AZIN1 cDNA is 1,353 bp in length, encoding a 450 amino acid protein with a molecular weight of 50 kDa. Out of all tissues examined, AZIN1 expression was highest in the adrenal gland and lowest in breast muscle. There was also a high expression of AZIN1 in the cerebellum and isthmus of oviduct. With follicular development, AZIN1 gene expression gradually increased, and its expression in F1 was significantly higher than in F5 (P<0.05). AZIN1 expression was also significantly higher in the POF1 than in the other follicles (P<0.05), and there was a low mRNA expression of AZIN1 in atretic follicles. The results of AZIN1 expression profiling in ovarian follicles suggest that AZIN1 may play an important role in the progression of follicular development, potentially through regulating polyamine levels.

  16. Chinese goose (Anser cygnoides) CD8a: cloning, tissue distribution and immunobiological in splenic mononuclear cells.

    PubMed

    Zhao, Qiurong; Liu, Fei; Chen, Shun; Yan, Xiaoling; Qi, Yulin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Chen, Xiaoyue; Cheng, Anchun

    2013-10-25

    CD8 molecule is a cell membrane glycoprotein, which plays an important role in cell-mediated immunity. Here, we identified Chinese goose CD8α (goCD8α) gene for the first time. The full-length cDNA of goCD8α is 1459bp in length and contains a 711bp open reading frame. Phylogenetic analysis shows that the waterfowl CD8α formed a monophyletic group. Semi-quantitative RT-PCR analysis showed that transcripts of goCD8α mRNA were high in the immune-related organs and mucosal immune system in gosling, and high in thymus and spleen comparing to other immune-related tissues in goose. The obvious increase of CD8α expression was observed in spleen of acute new type gosling viral enteritis virus (NGVEV) infected bird, while the increase of CD8α were observed in the thymus, bursa of fabricius, and cecum of chronic infected bird. The CD8α mRNA transcription level in spleen mononuclear cells was significantly up-regulated when stimulated by phytohemagglutinin, but not by lipopolysaccharide in vitro.

  17. Melatonin receptors in a pleuronectiform species, Solea senegalensis: Cloning, tissue expression, day-night and seasonal variations.

    PubMed

    Confente, Francesca; Rendón, María Carmen; Besseau, Laurence; Falcón, Jack; Muñoz-Cueto, José A

    2010-06-01

    Melatonin receptors are expressed in neural and peripheral tissues and mediate melatonin actions on the synchronization of circadian and circannual rhythms. In this study we have cloned three melatonin receptor subtypes (MT1, MT2 and Mel1c) in the Senegalese sole and analyzed their central and peripheral tissue distribution. The full-length MT1 (1452 nt), MT2 (1728 nt) and Mel1c (1980 nt) cDNAs encode different proteins of 345, 373, 355 amino acids, respectively. They were mainly expressed in retina, brain and pituitary, but MT1 was also expressed in gill, liver, intestine, kidney, spleen, heart and skin. At peripheral level, MT2 expression was only evident in gill, kidney and skin whereas Mel1c expression was restricted to the muscle and skin. This pattern of expression was not markedly different between sexes or among the times of day analyzed. The real-time quantitative PCR analyses showed that MT1 displayed higher expression at night than during the day in the retina and optic tectum. Seasonal MT1 expression was characterized by higher mRNA levels in spring and autumn equinoxes for the retina, and in winter and summer solstices for the optic tectum. An almost similar expression profile was found for MT2, but differences were less conspicuous. No day-night differences in MT1 and MT2 expression were observed in the pituitary but a seasonal variation was detected, being mRNA levels higher in summer for both receptors. Mel1c expression did not exhibit significant day-night variation in retina and optic tectum but showed seasonal variations, with higher transcript levels in summer (optic tectum) and autumn (retina). Our results suggest that day-night and seasonal variations in melatonin receptor expression could also be mediating circadian and circannual rhythms in sole.

  18. Tissue expression analysis, cloning and characterization of the 5'-regulatory region of the bovine FABP3 gene.

    PubMed

    Li, Anning; Wu, Lijuan; Wang, Xiaoyu; Xin, Yaping; Zan, Linsen

    2016-09-01

    Fatty acid binding protein 3 (FABP3) is a member of the FABP family which bind fatty acids and have an important role in fatty acid metabolism. A large number of studies have shown that the genetic polymorphisms of FABP3 are positively correlated with intramuscular fat (IMF) content in domestic animals, however, the function and transcriptional characteristics of FABP3 in cattle remain unclear. Real-time PCR analysis revealed that bovine FABP3 was highly expressed in cardiac tissue. The 5'-regulatory region of bovine FABP3 was cloned and its transcription initiation sites were identified. Sequence analysis showed that many transcriptional factor binding sites including TATA-box and CCAAT-box were present on the 5'-flanking region of bovine FABP3, and four CpG islands were found on nucleotides from -891 to +118. Seven serial deletion constructs of the 5'-regulatory region evaluated in dual-luciferase reporter assay indicated that its core promoter was 384 base pairs upstream from the transcription initiation site. The transcriptional factor binding sites RXRα, KLF15, CREB and Sp1 were conserved in the core promoter of cattle, sheep, pigs and dogs. These results provide further understanding of the function and regulation mechanism of bovine FABP3. PMID:27270359

  19. cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/Myeloid Leukemia Factor 2 (MLF2)

    SciTech Connect

    Kuefer, M.U.; Valentine, V.; Behm, F.G.

    1996-07-15

    A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, and MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements. 19 refs., 2 figs.

  20. Molecular cloning and characterization of amh, dax1 and cyp19a1a genes and their response to 17α-methyltestosterone in Pengze crucian carp.

    PubMed

    Li, Meng; Wang, Lihong; Wang, Houpeng; Liang, Hongwei; Zheng, Yao; Qin, Fang; Liu, Shaozhen; Zhang, Yingying; Wang, Zaizhao

    2013-05-01

    The proteins encoded by amh, dax1 and cyp19a1a play important roles in gonad differentiation. Their functions have been far less studied in teleosts. In this study, the full-length cDNAs of amh, dax1 and cyp19a1a were cloned and characterized in a triploid gynogenic fish, the Pengze crucian carp. Their expression profilings in juvenile development, adult tissues and juveniles exposed to 100 ng/L 17α-methyltestosterone (MT) were investigated. Results showed that their putative proteins shared high identities to their counterparts in cyprinid fish species, respectively. The tissue distribution results indicated that amh and cyp19a1a were predominantly expressed in the ovary and dax1 was dominantly expressed in the liver. Gene profiling in the developmental stages showed that all the three target genes had a consistent highest expression at 48 days post hatching (dph). The period of 48 dph appeared to be a key time during the process of the gonad development of Pengze crucian carp. 100 ng/L MT significantly increased the mRNA expression of amh at 2- and 4-week exposures and enhanced dax1 and cyp19a1a at 6-week exposure. The present study indicated that MT could influence the gonad development in Pengze crucian carp by disturbing sex-differentiation associated gene expression. Furthermore, the present study will be of great significance to broaden the understanding of molecular mechanisms of the physiological processes of reproduction in fish.

  1. Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis.

    PubMed Central

    Olsson, A; Hagström, T; Nilsson, B; Uhlén, M; Gatenbeck, S

    1985-01-01

    The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000. Images PMID:3923928

  2. Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.).

    PubMed

    He, MeiJing; Yang, XinLei; Cui, ShunLi; Mu, GuoJun; Hou, MingYu; Chen, HuanYing; Liu, LiFeng

    2015-08-15

    Annexin, Ca(2+) or phospholipid binding proteins, with many family members are distributed throughout all tissues during plant growth and development. Annexins participate in a number of physiological processes, such as exocytosis, cell elongation, nodule formation in legumes, maturation and stress response. Six different full-length cDNAs and two partial-length cDNAs of peanut, (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, AnnAh7, AnnAh4 and AnnAh8) encoding annexin proteins, were isolated and characterized using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins were 36.0kDa with acidic pIs of 5.97-8.81. ANNAh1, ANNAh2, ANNAh3, ANNAh5, ANNAh6 and ANNAh7 shared sequence similarity from 35.76 to 66.35% at amino acid level. Phylogenetic analysis revealed their evolutionary relationships with corresponding orthologous sequences in soybean and deduced proteins in various plant species. Real-time quantitative assays indicated that these genes were differentially expressed in various organs. Transcript level analysis for six annexin genes under stress conditions showed that these genes were regulated by drought, salinity, heavy metal stress, low temperature and hormone. Additionally, the prediction of cis-regulatory element suggested that different cis-responsive elements including stress- and hormone-responsive-related elements could respond to various stress conditions. These results indicated that members of AnnAhs family may play important roles in the adaptation of peanut to various environmental stresses.

  3. Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.).

    PubMed

    He, MeiJing; Yang, XinLei; Cui, ShunLi; Mu, GuoJun; Hou, MingYu; Chen, HuanYing; Liu, LiFeng

    2015-08-15

    Annexin, Ca(2+) or phospholipid binding proteins, with many family members are distributed throughout all tissues during plant growth and development. Annexins participate in a number of physiological processes, such as exocytosis, cell elongation, nodule formation in legumes, maturation and stress response. Six different full-length cDNAs and two partial-length cDNAs of peanut, (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, AnnAh7, AnnAh4 and AnnAh8) encoding annexin proteins, were isolated and characterized using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins were 36.0kDa with acidic pIs of 5.97-8.81. ANNAh1, ANNAh2, ANNAh3, ANNAh5, ANNAh6 and ANNAh7 shared sequence similarity from 35.76 to 66.35% at amino acid level. Phylogenetic analysis revealed their evolutionary relationships with corresponding orthologous sequences in soybean and deduced proteins in various plant species. Real-time quantitative assays indicated that these genes were differentially expressed in various organs. Transcript level analysis for six annexin genes under stress conditions showed that these genes were regulated by drought, salinity, heavy metal stress, low temperature and hormone. Additionally, the prediction of cis-regulatory element suggested that different cis-responsive elements including stress- and hormone-responsive-related elements could respond to various stress conditions. These results indicated that members of AnnAhs family may play important roles in the adaptation of peanut to various environmental stresses. PMID:25958350

  4. Transfection of Arabidopsis protoplasts with a Plum pox virus (PPV) infectious clone for studying early molecular events associated with PPV infection.

    PubMed

    Raghupathy, Mohan B; Griffiths, Jonathan S; Stobbs, Lorne W; Brown, Daniel C W; Brandle, James E; Wang, Aiming

    2006-09-01

    The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection. PMID:16777241

  5. Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

    PubMed

    Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

    2013-11-01

    Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

  6. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    SciTech Connect

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J. )

    1988-10-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 {times} 10{sup 6} recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria.

  7. Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

    PubMed

    Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

    2013-11-01

    Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate. PMID:24199859

  8. Molecular cloning and expression analysis of GhLOF genes in upland cotton (Gossypium hirsutum L.).

    PubMed

    Dai, T C; Wang, Z M

    2015-05-04

    Shoot branching, i.e., the timing and position of shoot growth, determines to a large extend the pattern of plant architecture, and is the result of the integration of a plant's genetic background and environmental cues. Many genes that are involved in the formation and outgrowth of axillary buds have been cloned, but the exact mechanism is still unclear. Branching pattern is an important agronomic trait in many crops, including cotton. In the present study, we cloned four genes from cotton, and designated them as GhLOF1/2/3/4. Sequence analysis revealed that all four genes shared conserved protein domains with LATERAL ORGAN FUSION (LOF) from Arabidopsis and TRIFOLIATE (Tf) from tomato. Phylogenetic analysis revealed that GhLOF3 and GhLOF4 were close to Tf because of their similar expression patterns, whereas GhLOF1 and GhLOF2 were differentially expressed.

  9. Molecular Cloning and Heterologous Expression of the Dehydrophos Biosynthetic Gene Cluster

    PubMed Central

    Circello, Benjamin T.; Eliot, Andrew C.; Lee, Jin-Hee; van der Donk, Wilfred A.; Metcalf, William W.

    2010-01-01

    Summary Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of Streptomyces lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments. PMID:20416511

  10. Molecular cloning and characterization of two novel cellulase genes from the mollusc Ampullaria crossean.

    PubMed

    Guo, Rui; Ding, Ming; Zhang, Si-Liang; Xu, Gen-Jun; Zhao, Fu-Kun

    2008-02-01

    Cellulase genes have been reported not only from fungi, bacteria and plant, but also from some invertebrate animals. Here, two cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) genes, eg27I and eg27II, were cloned from the freshwater snail Ampullaria crossean cDNA using degenerate primers. The nucleotide sequences of the two genes shared 94.5% identity. The open reading frames of both genes consisted of 588 bp, encoding 195 amino acids. Both EG27I and EG27II belong to the glycoside hydrolase family 45, and each lacks a carbohydrate-binding module. The presence of introns demonstrated a eukaryotic origin of the EG27 gene, and, in addition, successful cloning of EG27 cDNA supported endogenous production of EG27 cellulase by Ampullaria crossean. Investigation of the EG27 cDNA from A. crossean will provide further information on GHF45 cellulases.

  11. Molecular cloning, DNA structure and expression of the Escherichia coli D-xylose isomerase.

    PubMed Central

    Briggs, K A; Lancashire, W E; Hartley, B S

    1984-01-01

    The D-xylose isomerase (EC 5.3.1.5) gene from Escherichia coli was cloned and isolated by complementation of an isomerase-deficient E. coli strain. The insert containing the gene was restriction mapped and further subcloning located the gene in a 1.6-kb Bg/II fragment. This fragment was sequenced by the chain termination method, and showed the gene to be 1002 bp in size. The Bg/II fragment was cloned into a yeast expression vector utilising the CYCl yeast promoter. This construct allowed expression in E. coli grown on xylose but not glucose suggesting that the yeast promoter is responding to the E. coli catabolite repression system. No expression was detected in yeast from this construct and this is discussed in terms of the upstream region in the E. coli insert with suggestions of how improved constructs may permit achievement of the goal of a xylose-fermenting yeast. PMID:6325179

  12. Molecular cloning and expression of a human heat shock factor, HSF1

    SciTech Connect

    Rabindran, S.K.; Giorgi, G.; Clos, J.; Wu, C. )

    1991-08-15

    Human cells respond to heat stress by inducing the binding of a preexisting transcriptional activator (heat shock factor, HSF) to DNA. The authors isolated recombinant DNA clones for a human cDNA fragment. The human HSF1 probe was produced by the PCR with primers deduced from conserved amino acids in the Drosophila and yeast HSF sequences. The human HSF1 mRNA is constitutively expressed in HeLa cells under nonshock conditions and encodes a protein with four conserved leucine zipper motifs. Like its counterpart in Drosophila, human HSF1 produced in Escherichia coli in the absence of heat shock is active as a DNA binding transcription factor, suggesting that the intrinsic activity of HSF is under negative control in human cells. Surprisingly, an independently isolated human HSF clone, HSF2, is related to but significantly different from HSF.

  13. Molecular cloning and in vitro expression of a silent phenoxazinone synthase gene from Streptomyces lividans.

    PubMed

    Madu, A C; Jones, G H

    1989-12-14

    Phenoxazinone synthase (PHS) catalyzes a step in actinomycin D biosynthesis in Streptomyces antibioticus. Two sequences from Streptomyces lividans that hybridize to the phs gene of S. antibioticus have been cloned in Escherichia coli K-12 using the plasmid pBR322. Although there was some similarity in the restriction maps of the two cloned fragments, neither insert appeared to be a direct subset of the other nor of the S. antibioticus phs gene. In vitro expression studies, in a streptomycete coupled transcription-translation system, showed that a 3.98-kb SphI fragment encoded a PHS-related protein. These observations provide additional support for the existence of silent genes for antibiotic production in streptomycetes.

  14. Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen.

    PubMed

    Strzalka, Wojciech; Ziemienowicz, Alicja

    2007-02-01

    A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.

  15. Molecular cloning and characterization of human pyruvate dehydrogenase. beta. subunit gene

    SciTech Connect

    Koike, Kichiko; Urata, Yoshishige; Koike, Masahiko )

    1990-08-01

    A genomic clone encompassing the entire gene for the human pyruvate dehydrogenase {beta} subunit (PDH{beta}) has been isolated by screening a leukocyte genomic library with a nick-translated human foreskin fibroblast PDH{beta} cDNA probe. The 18-kilobase clone was characterized by restriction enzyme analysis, extensive DNA sequencing, and primer-extension analysis. The PDH{beta} structural gene is composed of 10 exons and 9 introns. All intron-exon splice junctions follow the GT/AG rule. The Alu family was found in introns 2 and 8. The 5{prime} flanking region of the PDH{beta} gene contains a CAAT consensus promoter sequence but no TATA sequence. Primer-extension analysis indicated the PDH{beta} gene transcription start site is an adenine residue located 132 bases upstream from the initiation codon in exon 1.

  16. Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride

    PubMed Central

    Matroudi, S.; Zamani, M.R.; Motallebi, M.

    2008-01-01

    In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli. PMID:24031242

  17. Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.

    PubMed Central

    Corey, E J; Matsuda, S P; Bartel, B

    1994-01-01

    We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter. Images PMID:8134375

  18. Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase.

    PubMed

    Uchimura, K; Muramatsu, H; Kadomatsu, K; Fan, Q W; Kurosawa, N; Mitsuoka, C; Kannagi, R; Habuchi, O; Muramatsu, T

    1998-08-28

    We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcbeta1-3Galbeta1-4GlcNAc. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcalpha2-3Galbeta1-4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galbeta1-4(Fucalpha1-3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.

  19. Nitrocellulose immunoblotting for identification and molecular gene cloning of Eimeria maxima antigens that stimulate lymphocyte proliferation.

    PubMed Central

    Bumstead, J M; Dunn, P P; Tomley, F M

    1995-01-01

    An immunoblotting technique was used to identify lymphostimulatory antigens within sized polypeptide fractions of Eimeria maxima sporozoites. Six fractions contained polypeptides that specifically stimulated the proliferation of immune lymphocytes in an in vitro assay, and polyclonal antisera were made in rabbits against these fractions. cDNA clones, isolated with antisera against a lymphostimulatory fraction of around 70 kDa, were found to encode four different antigens including a classical hsp70, a molecule homologous to an endoplasmic reticulum chaperonin (BiP/GRP), and a calcium-dependent serine/threonine protein kinase that appears homologous to a recently described molecule from Plasmodium falciparum. The protein kinase cDNA clone was overexpressed in Escherichia coli, and the recombinant antigen was found to induce both antibody and lymphoproliferative responses in chickens when administered subcutaneously. Thus, immunoblotting, in combination with in vitro lymphoproliferation assays, can be used as an initial screen for the identification of lymphostimulatory antigens from a complex pool of polypeptides, and a combination of cDNA cloning, expression, and immunization allows assessment of the lymphostimulatory activity of individual polypeptides. These studies should facilitate further evaluation of antigens that are potential candidates for inclusion in a recombinant vaccine against poultry coccidiosis. PMID:8548529

  20. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

    PubMed Central

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus. PMID:25174962

  1. Molecular cloning, bioinformatics analysis and functional characterization of HWTX-XI toxin superfamily from the spider Ornithoctonus huwena.

    PubMed

    Jiang, Liping; Deng, Meichun; Duan, Zhigui; Tang, Xing; Liang, Songping

    2014-04-01

    Spider venom contains a very valuable repertoire of natural resources to discover novel components for molecular diversity analyses and therapeutic applications. In this study, HWTX-XI toxins from the spider venom glands of Ornithoctonus huwena which are Kunitz-type toxins (KTTs) and were directly cloned, analyzed and functionally characterized. To date, the HWTX-XI superfamily consists of 38 members deduced from 121 high-quality expressed sequence tags, which is the largest spider KTT superfamily with significant molecular diversity mainly resulted from cDNA tandem repeats as well as focal hypermutation. Among them, HW11c40 and HW11c50 may be intermediate variants between native Kunitz toxins and sub-Kunitz toxins based on evolutionary analyses. In order to elucidate their biological activities, recombinant HW11c4, HW11c24, HW11c27 and HW11c39 were successfully expressed, further purified and functionally characterized. Both HW11c4 and HW11c27 display inhibitory activities against trypsin, chymotrypsin and kallikrein. Moreover, HW11c4 is also an inhibitor relatively specific for Kv1.1 channels. HW11c24 and HW11c39 are found to be inactive on chymotrysin, trypsin, kallikrein, thrombin and ion channels. These findings provide molecular evidence for toxin diversification of the HWTX-XI superfamily and useful molecular templates of serine protease inhibitors and ion channel blockers for the development of potentially clinical applications.

  2. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

    PubMed

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus. PMID:25174962

  3. Molecular cloning, sequencing, and distribution of feline GnRH receptor (GnRHR) and resequencing of canine GnRHR.

    PubMed

    Samoylov, Alexandre M; Napier, India D; Morrison, Nancy E; Martin, Douglas R; Cox, Nancy R; Samoylova, Tatiana I

    2015-01-15

    GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species.

  4. Molecular cloning and sequence analysis of selenoprotein W gene and its mRNA expression patterns in response to metabolic status and cadmium exposure in goldfish, Carassius auratus.

    PubMed

    Chen, Wenbo; Zhang, Zhen; Dong, Haiyan; Jiang, Xiaoxue

    2015-06-01

    Selenoprotein W (SelW) is a low molecular weight and selenocysteine containing protein with redox activity involved in the antioxidant response. In the present study, the full-length cDNA of goldfish (Carassius auratus) selenoprotein W (gfSelW) was successfully cloned from the liver tissue by rapid amplification of cDNA ends technique. The obtained gfSelW cDNA was 730 bp long with a 79 bp 5'-untranslated region (UTR), a 390 bp 3'-UTR containing the consensus polyadenylation signal AATAAA and a 261 bp open reading frame coding a protein of 86 amino acid residues. gfSelW mRNA was observed in all regions of brain and peripheral tissues by semi-quantitative RT-PCR, and the most abundant was detected in testis. After fasting for 1 week, gfSelW mRNA expression levels were significantly decreased compared to the fed group in hypothalamus and liver. After refeeding for 7 days, gfSelW mRNA expression levels were increased back. Furthermore, the mRNA expressions of gfSelW in hypothalamus and liver were varied in periprandial changes and significantly up-regulated after meal 2 h and 4 h, respectively. With cadmium exposure for 24 h, gfSelW mRNA expression levels in gill and leucocytes were significantly decreased at different cadmium concentrations changing from 0.5 ppm to 10 ppm. However, the gfSelW mRNA expression level was sharply increased in liver, relatively to the control about 4.98-fold at 0.5 ppm. The results in this study provide molecular characterization of SelW in goldfish and imply that SelW mRNA expression may be associated with metabolic status and oxidative stress and regulated by metabolic factors and cadmium in fish.

  5. Molecular cloning and characterization of the full-length Hsp90 gene from Matricaria recutita.

    PubMed

    Ling, S P; Su, S S; Zhang, H M; Zhang, X S; Liu, X Y; Pan, G F; Yuan, Y

    2014-01-01

    Heat shock protein 90 (Hsp90) is one of the most abundant and conserved chaperone proteins and plays important roles in plant growth and responses to environmental stimuli. However, little is known regarding the sequence and function of Hsp90s in Matricaria recutita. In the present study, we cloned the full-length cDNA sequence of the hsp90 gene from this species. Using rapid amplification of cDNA ends technologies with 2 degenerate primers that were designed based on the hsp90 gene sequence from other members of Asteraceae, we isolated and characterized an Hsp90 homolog gene from M. recutita (Mr-Hsp90). The full-length Mr-hsp90 cDNA sequence, containing 2097 base pairs, encodes a protein of 698 amino acids. Based on amino acid sequence identity, Mr-Hsp90 showed high similarity to other cloned Hsp90 proteins. The Mr-Hsp90 protein was closely clustered with the Lactuca sativa in a phylogenetic tree. These results indicate that the cloned sequence of Mr-Hsp90 is a member of the Hsp90 family, which is reported for the first time in M. recutita. Next, we conducted a salt stress experiment to determine the protein's function under salt stress conditions. Survival of chamomile seedlings subjected to heat-shock pretreatment was significantly increased compared with groups that had not undergone heat-shock pretreatment in a salt stress environment. This indicates that Mr-Hsp90 plays an important role in the salt resistance of chamomile seedlings. PMID:25526220

  6. Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

    SciTech Connect

    Hondred, D. ); Hanson, A.D. Univ. de Montreal, Quebec )

    1990-09-01

    In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

  7. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    SciTech Connect

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  8. Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa.

    PubMed Central

    Paietta, J V

    1989-01-01

    The ars-1+ gene of Neurospora crassa encodes the enzyme arylsulfatase. ars-1+ is in a group of highly regulated sulfur-related structural genes that are expressed under conditions of sulfur limitation and are under coordinate control of the cys-3+ and scon+ regulatory genes. The ars-1+ gene was cloned by chromosome walking from the qa gene cluster, using a lambda library. Cotransformation of an N. crassa ars-1 mutant with the isolated lambda clones and the benomyl resistance gene, followed by assay for arylsulfatase activity, was used to screen for the ars-1+ gene. Further confirmation that the cloned segment mapped to the ars-1+ locus was obtained by restriction-fragment-length polymorphism analysis. Northern (RNA) blot analysis showed that the ars-1+ gene was transcribed to give an mRNA of 2.3 kilobases. In wild-type cells, the ars-1+ transcript was abundant under sulfur-derepressing conditions but absent under repressing conditions. Time course analysis showed that the appearance of ars-1+ message in sulfur-derepressed cultures paralleled the appearance of arylsulfatase enzyme activity. In addition, transcription of ars-1+ was detected only under derepressing conditions in a nuclear transcription assay. In a cys-3 regulatory mutant that was unable to synthesize arylsulfatase (or other sulfur-controlled enzymes), there was no ars-1+ transcript under repressing or derepressing conditions. In a temperature-sensitive cys-3 mutant, the ars-1+ transcript was present only at the permissive growth temperature and under sulfur derepression. A negative regulatory mutant, sconc, displayed both constitutive expression of arylsulfatase enzyme activity and content of ars-1+ message. Images PMID:2528685

  9. Molecular cloning and expression analysis of cDNAs encoding androgenic gland hormone precursors from two porcellionidae species, Porcellio scaber and P. dilatatus.

    PubMed

    Ohira, Tsuyoshi; Hasegawa, Yuriko; Tominaga, Satoshi; Okuno, Atsuro; Nagasawa, Hiromichi

    2003-01-01

    Male sexual characteristics in Crustacea are induced by androgenic gland hormone (AGH), which is produced by the male-specific androgenic gland. Recently, AGH in the terrestrial isopod Armadillidium vulgare was characterized and its cDNA cloned, the first example in which the structure of AGH was elucidated. We report here the molecular cloning of cDNAs encoding AGH precursors from two additional terrestrial isopods, Porcellio scaber and P. dilatatus. cDNA fragments encoding Porcellio scaber AGH (Pos-AGH) and P. dilatatus AGH (Pod-AGH) were amplified by RT-PCR using degenerate oligonucleotide primers designed based on the amino acid sequence of A. vulgare AGH (Arv-AGH). Subsequently, full length cDNAs were obtained by 5'- and 3'-RACE. Both AGH precursors consisted of a signal peptide, B chain, C peptide and A chain, and exhibited the same organization as that of Arv-AGH. The amino acid sequences of the A and B chains, which comprise mature AGH peptide, were highly conserved among the three species, while that of the C peptide showed only low sequence similarity. In Northern blot analysis, each cDNA fragment used as a probe specifically hybridized with a single band (0.75 kb) in mRNA extracted from whole male reproductive organs. In analysis of the tissue-specific gene expression of these two AGHs by RT-PCR, it was revealed that both AGH transcripts were detected only in cDNA synthesized using total RNA from the testis carrying the androgenic glands, but not in that from testis only, seminal vesicle, vas deferens, or hepatopancreas. PMID:12560604

  10. Molecular cloning and expression analysis of cDNAs encoding androgenic gland hormone precursors from two porcellionidae species, Porcellio scaber and P. dilatatus.

    PubMed

    Ohira, Tsuyoshi; Hasegawa, Yuriko; Tominaga, Satoshi; Okuno, Atsuro; Nagasawa, Hiromichi

    2003-01-01

    Male sexual characteristics in Crustacea are induced by androgenic gland hormone (AGH), which is produced by the male-specific androgenic gland. Recently, AGH in the terrestrial isopod Armadillidium vulgare was characterized and its cDNA cloned, the first example in which the structure of AGH was elucidated. We report here the molecular cloning of cDNAs encoding AGH precursors from two additional terrestrial isopods, Porcellio scaber and P. dilatatus. cDNA fragments encoding Porcellio scaber AGH (Pos-AGH) and P. dilatatus AGH (Pod-AGH) were amplified by RT-PCR using degenerate oligonucleotide primers designed based on the amino acid sequence of A. vulgare AGH (Arv-AGH). Subsequently, full length cDNAs were obtained by 5'- and 3'-RACE. Both AGH precursors consisted of a signal peptide, B chain, C peptide and A chain, and exhibited the same organization as that of Arv-AGH. The amino acid sequences of the A and B chains, which comprise mature AGH peptide, were highly conserved among the three species, while that of the C peptide showed only low sequence similarity. In Northern blot analysis, each cDNA fragment used as a probe specifically hybridized with a single band (0.75 kb) in mRNA extracted from whole male reproductive organs. In analysis of the tissue-specific gene expression of these two AGHs by RT-PCR, it was revealed that both AGH transcripts were detected only in cDNA synthesized using total RNA from the testis carrying the androgenic glands, but not in that from testis only, seminal vesicle, vas deferens, or hepatopancreas.

  11. Molecular cloning, characterization and expression analysis of tumor suppressor protein p53 from orange-spotted grouper, Epinephelus coioides in response to temperature stress.

    PubMed

    Qi, Zeng-Hua; Liu, Yu-Feng; Luo, Sheng-Wei; Chen, Chu-Xian; Liu, Yuan; Wang, Wei-Na

    2013-11-01

    The tumor suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. In the present study, the cDNA of p53 from the orange-spotted grouper (Epinephelus coioides) (Ec-p53) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Ec-p53 was of 1921 bp, including an open reading frame (ORF) of 1143 bp encoding a polypeptide of 380 amino acids with predicted molecular weight of 42.3 kDa and theoretical isoelectric point of 7.0. Quantitative real-time PCR (qRT-PCR) assays revealed that Ec-p53 was ubiquitously expressed in all the examined tissues but with high levels in intestine and liver of the orange-spotted grouper. In addition, we measured the DNA damage and apoptosis in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in grouper in conditions where the temperature was 15 ± 0.5 °C. Furthermore, qRT-PCR and western blot confirmed that low temperature stress induced upregulation of Ec-p53 in the mRNA and protein levels. These results suggest that low temperature-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively stimulate the expression of Ec-p53, which plays a critical role in immune defense and antioxidant responses.

  12. Molecular characterization of Staphylococcus aureus isolates causing skin and soft tissue infections in patients from Malakand, Pakistan.

    PubMed

    Madzgalla, S; Syed, M A; Khan, M A; Rehman, S S; Müller, E; Reissig, A; Ehricht, R; Monecke, S

    2016-09-01

    Comparatively few studies have been published describing Staphylococcus aureus/MRSA epidemiology in Central Asia including Pakistan. Here, we report the genotyping of Staphylococcus aureus strains (that include both methicillin-susceptible and methicillin-resistant Staphylococcus aureus) from community- and hospital-acquired skin and soft-tissue infections in a tertiary care hospital in the Malakand district of the Khyber Pakhtunkhwa Province of Pakistan. Forty-five isolates of Staphylococcus aureus were characterized by microarray hybridization. Twenty isolates (44 %) were MRSA, whereas 22 (49 %) were PVL-positive. Fourteen isolates (31 %) harboured both mecA and PVL genes. The dominant clones were CC121-MSSA (n = 15, 33 %) and the PVL-positive "Bengal Bay Clone" (ST772-MRSA-V; n = 13, 29 %). The PVL-positive CC8-MRSA-IV strain "USA300" was found once. The pandemic ST239-MRSA-III strain was absent, although it has previously been observed in Pakistan. These observations require a re-assessment of schemes for initial antibiotic therapy to cover MRSA and they emphasise the need for a rapid and non-molecular test for PVL.

  13. Molecular cloning, characterization and expression of a gene encoding phosphoketolase from Termitomyces clypeatus.

    PubMed

    Sarkar, Prabuddha; Roy, Amit

    2014-05-16

    A phosphoketolase (pk) gene from the fungus Termitomyces clypeatus (TC) was cloned and partially characterized. Oligonucleotide primers specific for the phosphoketolase gene (pk) were designed from the regions of homologies found in the primary structure of the enzyme from other fungal sources related to TC, using multiple sequence alignment technique. The cDNA of partial lengths were amplified, cloned and sequenced in three parts by 3' and 5' RACE and RT-PCR using these oligonucleotide primers. The full length ds cDNA was constructed next by joining these three partial cDNA sequences having appropriate overlapping regions using Overlap Extension PCR technique. The constructed full length cDNA exhibited an open reading frame of 2487 bases and 5' and 3' UTRs. The deduced amino acid sequence, which is of 828 amino acids, when analyzed with NCBI BLAST, showed high similarities with the phosphoketolase enzyme (Pk) superfamily with expected domains. The part of the TC genomic DNA comprising of the pk gene was also amplified, cloned and sequenced and was found to contain two introns of 68 and 74 bases that interrupt the pk reading frame. The coding region of pk cDNA was subcloned in pKM260 expression vector in correct frame and the protein was expressed in Escherichia coli BL21 (DE3) transformed with this recombinant expression plasmid. The recombinant protein purified by His-tag affinity chromatography indicated the presence of a protein of the expected size. In vivo expression studies of the gene in presence of different carbon sources indicated synthesis of Pk specific mRNA, as expected. Phylogenetic studies revealed a common ancestry of the fungal and bacterial Pk. The TC is known to secrete several industrially important enzymes involved in carbohydrate metabolism. However, the presence of Pk, a key enzyme in pentose metabolism, has not been demonstrated conclusively in this organism. Cloning, sequencing and expression study of this gene establishes the functioning

  14. [Molecular cloning of Tupaia belangeri chinensis neuropeptide Y and homology comparison with other analogues from primates].

    PubMed

    Dong, Li; Lv, Long-Bao; Lai, Ren

    2012-02-01

    Much attention has been payed to tree shrews for their close phylogenetic relationship with primates, small size, and short reproductive cycle. Especially, they are considered as excellent experiential animals for medicine or/and disease research. A nucleotide sequence encoding neuropeptide Y(NPY) precursor has been cloned from the cDNA library of Tupaia belangeri chinensis. Sequence alignment revealed that the sequence homology with primate NPY was up to 96.9%. The phylogenetic analysis based on NPY precursor sequence revealed that the tree shrew has a close relationship with primates.

  15. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein.

    PubMed

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Kim, Suk

    2016-03-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.

  16. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein

    PubMed Central

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon

    2016-01-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis. PMID:27051349

  17. Molecular cloning, nucleotide sequence and expression of a Sulfolobus solfataricus gene encoding a class II fumarase.

    PubMed

    Colombo, S; Grisa, M; Tortora, P; Vanoni, M

    1994-01-01

    Fumarase catalyzes the interconversion of L-malate and fumarate. A Sulfolobus solfataricus fumarase gene (fumC) was cloned and sequenced. Typical archaebacterial regulatory sites were identified in the region flanking the fumC open reading frame. The fumC gene encodes a protein of 438 amino acids (47,899 Da) which shows several significant similarities with class II fumarases from both eubacterial and eukariotic sources as well as with aspartases. S. solfataricus fumarase expressed in Escherichia coli retains enzymatic activity and its thermostability is comparable to that of S. solfataricus purified enzyme despite a 11 amino acid C-terminal deletion.

  18. Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus.

    PubMed

    Li, Xundong; Zhuo, Kan; Luo, Mei; Sun, Longhua; Liao, Jinling

    2011-06-01

    The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2-70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus. PMID:21371475

  19. Cloning, characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[alpha]pyrene exposure.

    PubMed

    Xu, Chaoqun; Pan, Luqing; Liu, Na; Wang, Lin; Miao, Jingjing

    2010-08-01

    Glutathione S-transferases (GSTs) are phase II enzymes involved in major detoxification reactions of xenobiotics in many organisms. In this study, a full-length cDNA of GST-pi was cloned from the gill of Venerupis philippinarum by rapid amplification of cDNA ends (RACE) method for the first time. The full-length cDNA of V. philippinarum GST-pi (denoted as VpGSTp) was 1142bp, with a 5' untranslated region (UTR) of 87bp, a 3' UTR of 438bp, and an open reading frame (ORF) of 618bp encoding a protein of 205 amino acid residues with an estimated molecular mass of 23.9kDa and an predicted isoelectric point (pI) of 7.9. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzyme belongs to the pi-class, and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. Tissue distribution analysis of the VpGSTp mRNA revealed that the GST-pi expression level was observed higher in gill, adductor muscle, mantle and foot while lower in digestive gland. Using quantitative real-time PCR, the dose- and time-related effects of benzo[alpha]pyrene (B[alpha]P) on VpGSTp mRNA expression were investigated in gills and digestive gland. The results showed that a time-dependant increase in the expression of VpGSTp was induced by B[alpha]P and appeared a good linear relationship with B[alpha]P concentrations. All these results suggested that GST-pi in bivalve had an antioxidant role and VpGSTp expression may be a useful biomarker candidate for monitoring environmental contaminants such as PAHs.

  20. Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

    NASA Astrophysics Data System (ADS)

    Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

    2015-03-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

  1. Antimicrobial susceptibility, virulence determinant carriage and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections.

    PubMed

    Yu, Fangyou; Liu, Yunling; Lv, Jinnan; Qi, Xiuqin; Lu, Chaohui; Ding, Yu; Li, Dan; Liu, Huanle; Wang, Liangxing

    2015-01-01

    A better understanding of the antimicrobial susceptibility, carriage of virulence determinants and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections (SSTIs) may provide further insights related to clinical outcomes with these infections. From January 2012 to September 2013, a total of 128 non-duplicate S. aureus isolates were recovered from patients with SSTIs. All 128 S. aureus SSTI isolates carried at least five virulence genes tested. Virulence genes detected among at least 70% of all tested isolates included hld (100%), hla (95.3%), icaA (96.9%), clf (99.2%), sdrC (79.7%), sdrD (70.3%), and sdrE (72.7%). The prevalence of MRSA isolates with 10 virulence genes tested (54.4%, 31/56) was significantly higher than that among MSSA isolates (35.2%, 25/71) (p<0.05). The positive rates of seb, sen, sem, sdrE and pvl among MRSA isolates were significantly higher than among MSSA isolates (p<0.05). ST7 and ST630 accounting for 10.9% were found to be the predominant STs. The most prevalent spa type was t091 (8.6%). MRSA-ST59-SCCmec IV was the most common clone (12.3%) among MRSA isolates whereas among MSSA isolates the dominant clone was MSSA-ST7 (15.5%). Six main clonal complexes (CCs) were found, including CC5 (52.3%), CC7 (11.7%), CC59 (8.6%), CC88 (6.3%), CC398 (4.7%), and CC121 (3.1%). A higher carriage of seb and sec was found among CC59 isolates. In comparison to CC5 and CC7 isolates, those with the highest carriage rates (>80.0%) of sdrC and sdrD, CC59 isolates had lower prevalence of these two virulence genes. All CC59 isolates were susceptible to gentamicin and trimethoprim/sulfamethoxazole, while CC5 and CC7 isolates had resistance rates to these two antimicrobials of 25.4% and 20.9%, and 40.0% and 40.0%, respectively. The resistance rates for tetracycline, clindamycin, and erythromycin among CC5 isolates were lower than among CC7 and CC59 isolates. In conclusion, the molecular typing of S. aureus SSTI

  2. Cloning Yeast Actin cDNA Leads to an Investigative Approach for the Molecular Biology Laboratory

    ERIC Educational Resources Information Center

    Black, Michael W.; Tuan, Alice; Jonasson, Erin

    2008-01-01

    The emergence of molecular tools in multiple disciplines has elevated the importance of undergraduate laboratory courses that train students in molecular biology techniques. Although it would also be desirable to provide students with opportunities to apply these techniques in an investigative manner, this is generally not possible in the…

  3. Molecular cloning and expression analysis of a novel BCCP subunit gene from Aleurites moluccana.

    PubMed

    Xuan, W Y; Zhang, Y; Liu, Z Q; Feng, D; Luo, M Y

    2015-01-01

    Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate. PMID:26345927

  4. Molecular cloning and characterization of the Dicer-like 2 gene from Brassica rapa.

    PubMed

    Yan, Fei; Peng, Jiejun; Lu, Yuwen; Lin, Lin; Zheng, Hongying; Chen, Hairu; Chen, Jianping; Adams, Michael J

    2009-07-01

    Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3' end of BrDCL2, clones with three different lengths of 3' untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain.

  5. Cloning and molecular analysis of poly(3-hydroxyalkanoate) biosynthesis genes in Pseudomonas aureofaciens.

    PubMed

    Nishikawa, Tomohiro; Ogawa, Keiko; Kohda, Ryoko; Zhixiong, Wang; Miyasaka, Hitoshi; Umeda, Fusako; Maeda, Isamu; Kawase, Masaya; Yagi, Kiyohito

    2002-02-01

    Pseudomonas aureofaciens grown on octanoate or gluconate synthesized medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To clone the PHA synthase gene(s) (phaC), the genomic library of P. aureofaciens was constructed using a cosmid vector. The recombinant cosmids that clone phaC were detected by the complementation with a PHA-negative mutant, P. putida GPp104. The resulting recombinant cosmid, named pVK6, contained a 13-kbp DNA insert. Genetic analysis of the pha locus in pVK6 revealed the presence of six ORFs, genes encoding two PHA synthases, 1 and 2 (phaC1 and phaC2), PHA depolymerase (phaZ), two PHA granule-associated proteins (phaF and phaI), and an unknown protein (phaD). The heterologous expression of pha genes from P. aureofaciens was confirmed. P. putida GPp104 regained the ability to accumulate PHA on introduction of pVK6. Wild-type strains P. oleovorans and P. fluorescens, which were unable to accumulate PHA when grown on gluconate, acquired the ability to accumulate PHA from gluconate when they possessed pVK6. PMID:11815858

  6. Molecular cloning and sequence determination of DA strain of Theiler's murine encephalomyelitis viruses.

    PubMed

    Ohara, Y; Stein, S; Fu, J L; Stillman, L; Klaman, L; Roos, R P

    1988-05-01

    Theiler's murine encephalomyelitis viruses (TMEV) belong to the Picornaviridae, and are divided into two subgroups. TO subgroup strains produce a persistent demyelinating central nervous system infection in mice, while GDVII subgroup strains cause acute polioencephalomyelitis. We generated three overlapping clones of the genome of DA strain, a member of TO subgroup. Sequence analysis revealed that the genome is 8093 nucleotides long with a poly(A) tail. The 5' noncoding region stretches from nucleotides 1 to 1065 and lacks a poly(C) tract. The open reading frame stretches from 1066 to 7968 and encodes 2301 amino acids. DA strain sequence is more closely related to members of the Cardiovirus genus than to members of other Picornavirus genera. Comparison with sequence of BeAn strain, another TO subgroup strain, showed that the P1 area has the greatest number of differences, while the noncoding regions are more well-conserved. The three overlapping clones will be important in recombinant infectious cDNA studies between strains of both subgroups.

  7. Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis.

    PubMed Central

    Charles, I G; Dougan, G; Pickard, D; Chatfield, S; Smith, M; Novotny, P; Morrissey, P; Fairweather, N F

    1989-01-01

    Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough. We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus. Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B. pertussis CN2992. Analysis of the DNA sequence reveals a G + C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor. In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon. At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination. A full-length clone of the gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host. The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E. coli. Images PMID:2542937

  8. Molecular characterization of tobacco sulfite reductase: enzyme purification, gene cloning, and gene expression analysis.

    PubMed

    Yonekura-Sakakibara, K; Ashikari, T; Tanaka, Y; Kusumi, T a; Hase, T

    1998-09-01

    A cDNA clone, NtSiR1, that encodes the precursor of ferredoxin-dependent sulfite reductase (Fd-SiR) has been isolated from a cDNA library of tobacco (Nicotiana tabacum cv. SR1). The identity of the cDNA was established by comparison of the purified protein and the predicted structure with the nucleotide sequence. The amino terminus of the purified enzyme was Thr62 of the precursor protein, and the mature region of NtSiR1 consisted of 632 amino acids. Tobacco Fd-SiR is 82, 77, and 48% identical with Fd-SiRs from Zea mays, Arabidopsis thaliana, and a cyanobacterium, respectively. Significant similarity was also found with Escherichia coli NADPH-SiR in the region involved in ligation of siroheme and the [4Fe-4S] cluster. On Northern blot analysis, a transcript of NtSiR1 was detected in leaves, stems, roots, and petals in similar amounts. We also isolated a genomic SiR clone named gNtSiR1. It consists of 8 exons and 7 introns. Genomic Southern blot analysis indicated that at least two SiR genes are present in the tobacco genome. PMID:9722674

  9. Molecular cloning of HSP70 in Mycoplasma ovipneumoniae and comparison with that of other mycoplasmas.

    PubMed

    Li, M; Ma, C J; Liu, X M; Zhao, D; Xu, Q C; Wang, Y J

    2011-05-10

    Mycoplasma ovipneumoniae, a bacterial species that specifically affects ovine and goat, is the cause of ovine infectious pleuropneumonia. We cloned, sequenced and analyzed heat shock protein 70 (HSP70) (dnaK) gene of M. ovipneumoniae. The full length open reading frame of the M. ovipneumoniae HSP70 gene consists of 1812 nucleotides, with a G+C content of 34.16%, encoding 604 amino acids. Comparative analysis with the HSP70 sequences of 15 Mycoplasma species revealed 59 to 87% DNA sequence identity, with an amino acid sequence identity range of 58 to 94%. M. ovipneumoniae and M. hyopneumoniae shared the highest DNA and amino acid sequence identity (87 and 94%, respectively). Based on phylogenetic analysis, both the DNA and amino acid identities of M. ovipneumoniae with other mycoplasmal HSP70 were correlated with the degree of relationship between the species. The C-terminus of the HSP70 was cloned into a bacterial expression vector and expressed in Escherichia coli cells. The recombinant C-terminal portion of HSP70 protein strongly reacted with convalescent sera from M. ovipneumoniae-infected sheep, based on an immunoblotting assay. This indicates that HSP70 is immunogenic in a natural M. ovipneumoniae infection and may be a relevant antigen for vaccine development.

  10. Molecular cloning of a cDNA encoding the human Sm-D autoantigen

    SciTech Connect

    Rokeach, L.A.; Haselby, J.A.; Hoch, S.O. )

    1988-07-01

    Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. The authors have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg){sub 9} repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen.

  11. Molecular cloning and sequence analysis of striped bass (Morone saxatilis) gonadotrophin-I and -II subunits.

    PubMed

    Hassin, S; Elizur, A; Zohar, Y

    1995-08-01

    Two types of cDNA, each encoding a different beta-subunit of striped bass (Morone saxatilis, Teleostei) gonadotrophins (GTH-I beta and GTH-II beta), as well as the glycoprotein alpha-subunit, were cloned by screening a striped bass pituitary cDNA library. The probes used for screening the library were cloned cDNA fragments, generated by PCR amplification of reverse-transcribed mRNA obtained from two pituitaries. The nucleotide sequences of the alpha-subunit, GTH-I beta and GTH-II beta are 626, 524 and 580 bases long, encoding peptides of 117, 120 and 147 amino acids respectively. Striped bass GTH-I beta and GTH-II beta share a sequence identity of 48% at the nucleic acid level, and 30% at the amino acid level. A cluster analysis of vertebrate pituitary glycoprotein beta-subunits suggests that teleost GTH-II beta is more closely related to tetrapod LH than to FSH. Administration of gonadotrophin-releasing hormone analogue ([D-Ala6,Pro9Net]-LHRH) to juvenile striped resulted in ten-, two- and fivefold increases in the expression of the alpha-subunit, GTH-I beta and GTH-II beta respectively. These results suggest that each of the GTH subunits is differentially regulated, and further corroborate the functional duality of teleost gonadotrophins.

  12. Molecular cloning and characterization of Hymenolepis diminuta alpha-tubulin gene.

    PubMed

    Mohajer-Maghari, Behrokh; Amini-Bavil-Olyaee, Samad; Webb, Rodney A; Coe, Imogen R

    2007-02-01

    To isolate a full-length alpha-tubulin cDNA from an eucestode, Hymenolepis diminuta, a lambda phage cDNA library was constructed. The alpha-tubulin gene was cloned, sequenced and characterized. The H. diminuta alpha-tubulin consisted of 450 amino acids. This protein contained putative sites for all posttranslational modifications as detyrosination/tyrosination at the carboxyl-terminal of protien, phosphorylation at residues R79 and K336, glycylation/glutamylation at residue G445 and acetylation at residue K40. Comparisons of H. diminuta alpha-tubulin with all full-length alpha-tubulin proteins revealed that H. diminuta alpha-tubulin possesses 10 distinctive residues, which are not found in any other alpha-tubulins. Phylogenetic analysis showed that H. diminuta alpha-tubulin has grouped in a separated branch adjacent eucestode and trematodes branch with 92% bootstrap value (1000 replicates). In conclusion, this is the first report of H. diminuta cDNA library construction, cloning and characterization of H. diminuta alpha-tubulin gene.

  13. Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin.

    PubMed

    Van Damme, E J; Kaku, H; Perini, F; Goldstein, I J; Peeters, B; Yagi, F; Decock, B; Peumans, W J

    1991-11-15

    Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.

  14. A molecular approach to identify active microbes in environmental eukaryote clone libraries.

    PubMed

    Stoeck, Thorsten; Zuendorf, Alexandra; Breiner, Hans-Werner; Behnke, Anke

    2007-02-01

    A rapid method for the simultaneous extraction of RNA and DNA from eukaryote plankton samples was developed in order to discriminate between indigenous active cells and signals from inactive or even dead organisms. The method was tested using samples from below the chemocline of an anoxic Danish fjord. The simple protocol yielded RNA and DNA of a purity suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR) and PCR, respectively. We constructed an rRNA-derived and an rDNA-derived clone library to assess the composition of the microeukaryote assemblage under study and to identify physiologically active constituents of the community. We retrieved nearly 600 protistan target clones, which grouped into 84 different phylotypes (98% sequence similarity). Of these phylotypes, 27% occurred in both libraries, 25% exclusively in the rRNA library, and 48% exclusively in the rDNA library. Both libraries revealed good correspondence of the general community composition in terms of higher taxonomic ranks. They were dominated by anaerobic ciliates and heterotrophic stramenopile flagellates thriving below the fjord's chemocline. The high abundance of these bacterivore organisms points out their role as a major trophic link in anoxic marine systems. A comparison of the two libraries identified phototrophic dinoflagellates, "uncultured marine alveolates group I," and different parasites, which were exclusively detected with the rDNA-derived library, as nonindigenous members of the anoxic microeukaryote community under study.

  15. Molecular cloning and expression of bovine kappa-casein in Escherichia coli

    SciTech Connect

    Kang, Y.C.; Richardson, T.

    1988-01-01

    A cDNA library was constructed using poly(A)/sup +/RNA from bovine mammary gland. This cDNA library of 6000 clones was screened employing colony hybridization using /sup 32/P-labelled oligonucleotide probes and restriction endonuclease mapping. The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method. The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein. The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76. The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively. Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein.

  16. Molecular cloning and sequencing analysis of the interferon β from Coturnix.

    PubMed

    Zheng, Bei; Chang, Wei-Shan

    2014-01-01

    One pair of primers was designed according to Gallus and Meleagris gallopavo interferon β (IFN-β) sequences published in GenBank. The primers and RNA extraction from the spleen of Coturnix were used to amplify Coturnix IFN-β cDNA by real-time polymerase chain reaction (RT-PCR). The product was cloned into pEasy-T1 vector. Evaluating recombinant plasmid by PCR and restriction enzyme digestion. Sequence the cloning sequences, comparing the sequencing results by NCBI. We successfully got a Coturnix IFN-β partial sequence. The sequence was subtyped and put to homologous analysis. The results suggested the homology of IFN-β gene of Coturnix and gene of Coturnix and chicken (88.7%), the homology of IFN-β gene of Coturnix and chicken (88.7%), the homology of IFN-β gene of Coturnix and Anas platyrhynchos (72.5%), the homology of IFN-β sequence registered in GenBank. The analysis of the genetic tree showed that the relationship of Coturnix and chicken IFN-β had a high homology. It can be seen that in this study we successfully got a partial sequence of IFN-β of quail. PMID:26155095

  17. Molecular cloning and characterization of the 78-kilodalton glucose-regulated protein of Trypanosoma cruzi.

    PubMed Central

    Tibbetts, R S; Kim, I Y; Olson, C L; Barthel, L M; Sullivan, M A; Winquist, A G; Miller, S D; Engman, D M

    1994-01-01

    The protozoan Trypanosoma cruzi is the etiologic agent of Chagas' disease, an illness responsible for morbidity and death among millions of Latin Americans. Mice also develop this disease when infected with T. cruzi and are a useful model organism for the study of parasite-specific immune responses. To identify immunogenic T. cruzi antigens, serum from an infected mouse was used to isolate clones from a T. cruzi epimastigote cDNA expression library. One of these clones was found to encode the 78-kDa glucose-regulated protein (grp78), the endoplasmic reticular member of the 70-kDa heat shock protein (hsp70) family. Like the mammalian and yeast grp78s, the T. cruzi protein contains an endoplasmic reticular leader peptide and a carboxyl-terminal endoplasmic reticular retention sequence. T. cruzi grp78 is encoded by a tandemly arranged family of three genes located on a chromosome of 1.6 Mb. The effects on grp78 expression of heat shock and tunicamycin treatment, the latter of which specifically stimulates mammalian grp78, were investigated. While the level of the grp78 protein remained constant under all circumstances, grp78 mRNA was unaffected by heat shock but induced fivefold by tunicamycin. Finally, we found that grp78 is the most immunogenic of the T. cruzi heat shock proteins we have characterized, reacting strongly in immunoblots with sera from infected mice. Images PMID:8188375

  18. Molecular cloning and functional expression of a sodium bicarbonate cotransporter from guinea-pig parotid glands.

    PubMed

    Koo, Na-Youn; Li, Jingchao; Hwang, Sung Min; Choi, Se-Young; Lee, Sung Joong; Oh, Seog-Bae; Kim, Joong-Soo; Lee, Jong Heun; Park, Kyungpyo

    2006-04-21

    We recently found that the concentration of HCO3- in guinea-pig saliva is very similar to that of human saliva; however, the entity that regulates HCO3- transport has not yet been fully characterized. In order to investigate the mechanism of HCO3- transport, we identified, cloned, and characterized a sodium bicarbonate (Na(+)/HCO3- cotransporter found in guinea-pig parotid glands (gpNBC1). The gpNBC1 gene encodes a 1079-amino acid protein that has 95% and 96% homology with human and mouse parotid NBC1, respectively. Oocytes expressing gpNBC1 were exposed to HCO3- or Na(+)-free solutions, which resulted in a marked change in membrane potentials (V(m)), suggesting that gpNBC1 is electrogenic. Likewise, a gpNBC1-mediated pH recovery was observed in gpNBC1 transfected human hepatoma cells; however, in the presence of 4, 4-diisothiocyanostilbene-2,2-disulfonic acid, a specific NBC1 inhibitor, such changes in V(m) and pH(i) were not observed. Together, the data show that the cloned guinea-pig gene is a functional, as well as sequence homologue of human NBC1. PMID:16513089

  19. Molecular cloning and expression of hardening-induced genes in Chlorella vulgaris C-27: the most abundant clone encodes a late embryogenesis abundant protein.

    PubMed

    Joh, T; Honjoh, K; Yoshimoto, M; Funabashi, J; Miyamoto, T; Hatano, S

    1995-01-01

    To investigate the effects of hardening on gene expression in Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27), a frost-hardy strain, 17 cDNA clones corresponding to hardening-induced Chlorella (hiC) genes were isolated by differential screening of a cDNA library from 6-h hardened cells. Northern blot analysis of transcripts of hiC genes showed that these genes are specifically induced by hardening and that their patterns of induction vary. Southern blots of genomic DNAs from two strains (Chlorella ellipsoidea Gerneck IAM C-102, chilling-sensitive; and C. vulgaris C-27, frost-hardy) of Chlorella indicated that ten hiC clones out of 17 hybridized only with DNA of strain C-27 and the other seven clones hybridized with DNA of both strains. However, of these seven clones, transcripts corresponding to six clones did not accumulate in strain C-102 at low temperatures. The sequence of a deduced protein encoded by the most abundant clone, hiC6, exhibited homology to sequences of Group III LEA (late embryogenesis abundant) proteins and had an amino-terminal amino acid sequence that was similar to the sequences of chloroplast transit peptides. PMID:7719632

  20. Visualizing molecular polar order in tissues via electromechanical coupling

    PubMed Central

    Denning, Denise; Alilat, Sofiane; Habelitz, Stefan; Fertala, Andrzej; Rodriguez, Brian J.

    2015-01-01

    Electron microscopy (EM) and atomic force microscopy (AFM) techniques have long been used to characterize collagen fibril ordering and alignment in connective tissues. These techniques, however, are unable to map collagen fibril polarity, i.e., the polar orientation that is directed from the amine to the carboxyl termini. Using a voltage modulated AFM-based technique called piezoresponse force microscopy (PFM), we show it is possible to visualize both the alignment of collagen fibrils within a tissue and the polar orientation of the fibrils with minimal sample preparation. We demonstrate the technique on rat tail tendon and porcine eye tissues in ambient conditions. In each sample, fibrils are arranged into domains whereby neighboring domains exhibit opposite polarizations, which in some cases extend to the individual fibrillar level. Uniform polarity has not been observed in any of the tissues studied. Evidence of anti-parallel ordering of the amine to carboxyl polarity in bundles of fibrils or in individual fibrils is found in all tissues, which has relevance for understanding mechanical and biofunctional properties and the formation of connective tissues. The technique can be applied to any biological material containing piezoelectric biopolymers or polysaccharides. PMID:22985991

  1. Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

    PubMed

    Troncoso-Ponce, M A; Rivoal, J; Venegas-Calerón, M; Dorion, S; Sánchez, R; Cejudo, F J; Garcés, R; Martínez-Force, E

    2012-07-01

    Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.

  2. Molecular cloning and biochemical characterization of the UDP-glucose: flavonoid 3-O-glucosyltransferase from Concord grape (Vitis labrusca).

    PubMed

    Hall, Dawn; Yuan, Xiao Xin; Murata, Jun; De Luca, Vincenzo

    2012-02-01

    Glucosylation of anthocyanidin substrates at the 3-O-position is crucial for the red pigmentation of grape berries and wine. The gene that encodes the enzyme involved in this reaction has been cloned from Vitis labrusca cv. Concord, heterologously expressed, and the recombinant enzyme (rVL3GT) was characterized. VL3GT has 96% amino acid sequence identity with Vitis vinifera VV3GT and groups phylogenetically with several other flavonoid 3-O-glycosyltransferases. In vitro substrate specificity studies and kinetic analyses of rVL3GT indicate that this enzyme preferentially glucosylates cyanidin as compared with quercetin. Crude protein extracts from several Concord grape tissues were assayed for glucosyltransferase activity with cyanidin and quercetin as acceptor substrates. A comparison of the VL3GT activities toward with these substrates showed that the 3GT enzyme activity is consistent with the expression of VL3GT in these tissues and is coincident with the biosynthesis of anthocyanins in both location and developmental stages. Enzyme activities in grape mesocarp, pre-veraison exocarp, leaf, flower bud, and flower tissues glucosylated quercetin but not cyanidin at high rates, suggesting the presence of additional enzymes which are able to glucosylate the 3-O-position of flavonols with higher specificity than anthocyanidins.

  3. Cloning, molecular modeling, and docking analysis of alkali-thermostable β-mannanase from Bacillus nealsonii PN-11.

    PubMed

    Chauhan, Prakram Singh; Tripathi, Satya Prakash; Sangamwar, Abhays T; Puri, Neena; Sharma, Prince; Gupta, Naveen

    2015-11-01

    An alkali-thermostable β-mannanase gene from Bacillus nealsonii PN-11 was cloned by functional screening of E. coli cells transformed with pSMART/HaeIII genomic library. The ORF encoding mannanase consisted of 1100 bp, corresponding to protein of 369 amino acids and has a catalytic domain belonging to glycoside hydrolase family 5. Cloned mannanase was smaller in size than the native mannanase by 10 kDa. This change in molecular mass could be because of difference in the glycosylation. The tertiary structure of the β-mannanase (MANPN11) was designed and it showed a classical (α/β) TIM-like barrel motif. Active site of MANPN11 was represented by 8 amino acid residues viz., Glu152, Trp189, His217, Tyr219, Glu247, Trp276, Trp285, and Tyr287. Model surface charge of MANPN11 predicted that surface near active site was mostly negative, and the opposite side was positive which might be responsible for the stability of the enzymes at high pH. Stability of MANPN11 at alkaline pH was further supported by the formation of a hydrophobic pocket near active site of the enzyme. To understand the ability of MANPN11 to bind with different substrates, docking studies were performed and found that mannopentose fitted properly into active site and form stable enzyme substrate complex.

  4. Molecular cloning and expression of a unique rabbit osteoclastic phosphotyrosyl phosphatase.

    PubMed Central

    Wu, L W; Baylink, D J; Lau, K H

    1996-01-01

    Tyrosyl phosphorylation plays an important regulatory role in osteoclast formation and activity. Phosphotyrosyl phosphatases (PTPs), in addition to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an important regulatory role in osteoclasts, we sought to clone an osteoclast-specific PTP. A putative full-length clone encoding a unique PTP (referred to as PTP-oc) was isolated from a 10-day-old rabbit osteoclastic cDNA library and sequenced. A single open reading frame predicts a protein with 405 amino acid residues containing a putative extracellular domain, a single transmembrane region, and an intracellular portion. PTP-oc is structurally unique in that, unlike most known transmembrane PTPs, it has a short extracellular region (eight residues), lacks a signal peptide proximal to the N-terminus, and contains only a single 'PTP catalytic domain'. The PTP catalytic domain shows 45-50% sequence identity with the catalytic domain of human HPTP beta and with the first catalytic domain of LCA. The PTP-oc gene exists as a single copy in the rabbit genome. The corresponding mRNA (3.8 kb) is expressed in osteoclasts but not in other bone-derived cells (e.g. osteoblasts and stromal cells). The 3.8 kb PTP-oc mRNA transcript was also expressed in the rabbit brain, kidney and spleen. However, the brain and kidney, but not osteoclasts or spleen, also expressed a larger transcript (6.5 kb). The PTP catalytic domain of PTP-oc was expressed as a GST-cPTP-oc fusion protein. In vitro phosphatase assays indicated that the purified fusion protein exhibited phosphatase activities at neutral pH values toward p-nitrophenyl phosphate, phosphotyrosyl Raytide, and phosphotyrosyl histone, whereas it had no appreciable activity toward phosphoseryl casein. In summary, we have: (a) cloned and sequenced the putative full-length cDNA of a unique PTP (PTP-oc) from rabbit osteoclasts; (b) shown that the mature 3.8 kb PTP-oc m

  5. Molecular basis for chloronium-mediated meroterpene cyclization: cloning, sequencing, and heterologous expression of the napyradiomycin biosynthetic gene cluster.

    PubMed

    Winter, Jaclyn M; Moffitt, Michelle C; Zazopoulos, Emmanuel; McAlpine, James B; Dorrestein, Pieter C; Moore, Bradley S

    2007-06-01

    Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.

  6. DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA.

    PubMed Central

    Toukdarian, A E; Lidstrom, M E

    1984-01-01

    DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight. Images PMID:6321444

  7. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

    PubMed

    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

  8. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

    PubMed

    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian. PMID:23915159

  9. An Entry/Gateway® cloning system for general expression of genes with molecular tags in Drosophila melanogaster

    PubMed Central

    Akbari, Omar S; Oliver, Daniel; Eyer, Katie; Pai, Chi-Yun

    2009-01-01

    Background Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway® vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells. Results The Entry/Gateway® system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway® based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei. Conclusion We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system

  10. Molecular cloning of the perilipin gene and its association with carcass and fat traits in Chinese ducks.

    PubMed

    Zhang, H L; Fan, H J; Liu, X L; Wu, Y; Hou, S S

    2013-01-01

    The perilipin (PLIN) gene is a candidate gene of carcass and fat traits in ducks. In order to study the molecular character of the PLIN gene and its function in different breeds of Chinese ducks, samples were obtained from the Chinese Academy of Agricultural Sciences Research Center for Birds, including 95 Peking ducks of the Z2 series, 91 Peking ducks of the Z4 series, 82 hybrid systems (Z2 x Z4), and 93 Cherry Valley ducks. We used RT-PCR and 3'-RACE to clone the duck PLIN gene, detect SNPs and analyze their associations with carcass and fat traits. A 2212-bp sequence was cloned with the complete coding region and a 3'-untranslated region. We found a nucleotide mutation (C → T) in exon 2 of the PLIN gene. There were no significant correlations between the 3 genotypes (CC, CT, TT) in breast muscle weight (BMW), leg muscle weight (LMW), subcutaneous fat weight (SFW), and intramuscular fat (IMF) in the Cherry Valley duck. The CC and CT genotypes had significant differences in carcass weight (CW), carcass net weight (CNW), and percentage of abdominal fat weight (AFW); there were significant differences in AFW and percentage of SFW. In Z4, there were no significant correlations between the 3 genotypes (TT, CC, and CT) in CW, BMW, LMW, SFW, AFW, the percentage of SFW and AFW, and IMF. CNW was significantly different between TT, CC, and CT genotypes. In Z2 x Z4, there were no significant correlations between the 3 genotypes in CW, BMW, LMW, SFW, AFW, the percentage of SFW and AFW, and IMF, while the CC and CT genotypes had significant differences in CNW. In Z2, there were no significant differences between the 3 genotypes in all traits. We deduced that the PLIN gene is a potential major gene. It is linked to a major gene affecting meat quality traits. This SNP has potential as a molecular marker for marker-assisted selection.

  11. Molecular cloning of the perilipin gene and its association with carcass and fat traits in Chinese ducks.

    PubMed

    Zhang, H L; Fan, H J; Liu, X L; Wu, Y; Hou, S S

    2013-01-01

    The perilipin (PLIN) gene is a candidate gene of carcass and fat traits in ducks. In order to study the molecular character of the PLIN gene and its function in different breeds of Chinese ducks, samples were obtained from the Chinese Academy of Agricultural Sciences Research Center for Birds, including 95 Peking ducks of the Z2 series, 91 Peking ducks of the Z4 series, 82 hybrid systems (Z2 x Z4), and 93 Cherry Valley ducks. We used RT-PCR and 3'-RACE to clone the duck PLIN gene, detect SNPs and analyze their associations with carcass and fat traits. A 2212-bp sequence was cloned with the complete coding region and a 3'-untranslated region. We found a nucleotide mutation (C → T) in exon 2 of the PLIN gene. There were no significant correlations between the 3 genotypes (CC, CT, TT) in breast muscle weight (BMW), leg muscle weight (LMW), subcutaneous fat weight (SFW), and intramuscular fat (IMF) in the Cherry Valley duck. The CC and CT genotypes had significant differences in carcass weight (CW), carcass net weight (CNW), and percentage of abdominal fat weight (AFW); there were significant differences in AFW and percentage of SFW. In Z4, there were no significant correlations between the 3 genotypes (TT, CC, and CT) in CW, BMW, LMW, SFW, AFW, the percentage of SFW and AFW, and IMF. CNW was significantly different between TT, CC, and CT genotypes. In Z2 x Z4, there were no significant correlations between the 3 genotypes in CW, BMW, LMW, SFW, AFW, the percentage of SFW and AFW, and IMF, while the CC and CT genotypes had significant differences in CNW. In Z2, there were no significant differences between the 3 genotypes in all traits. We deduced that the PLIN gene is a potential major gene. It is linked to a major gene affecting meat quality traits. This SNP has potential as a molecular marker for marker-assisted selection. PMID:23765965

  12. Molecular cloning, phylogenetic analysis and heat shock response of Babesia gibsoni heat shock protein 90.

    PubMed

    Yamasaki, Masahiro; Tsuboi, Yoshihiro; Taniyama, Yusuke; Uchida, Naohiro; Sato, Reeko; Nakamura, Kensuke; Ohta, Hiroshi; Takiguchi, Mitsuyoshi

    2016-09-01

    The Babesia gibsoni heat shock protein 90 (BgHSP90) gene was cloned and sequenced. The length of the gene was 2,610 bp with two introns. This gene was amplified from cDNA corresponding to full length coding sequence (CDS) with an open reading frame of 2,148 bp. A phylogenetic analysis of the CDS of HSP90 gene showed that B. gibsoni was most closely related to B. bovis and Babesia sp. BQ1/Lintan and lies within a phylogenetic cluster of protozoa. Moreover, mRNA transcription profile for BgHSP90 exposed to high temperature were examined by quantitative real-time reverse transcription-polymerase chain reaction. BgHSP90 levels were elevated when the parasites were incubated at 43°C for 1 hr. PMID:27149891

  13. Molecular cloning, genomic organization, and chromosomal localization of the human pancreatitis-associated protein (PAP) gene

    SciTech Connect

    Dusetti, N.J.; Frigerio, J.M.; Dagorn, J.C.; Iovanna, J.L. ); Fox, M.F.; Swallow, D.M. )

    1994-01-01

    Pancreatitis-associated protein (PAP) is a secretory pancreatic protein present in small amounts in normal pancreas and overexpressed during the acute phase of pancreatitis. In this paper, the authors describe the cloning, characterization, and chromosomal mapping of the human PAP gene. The gene spans 2748 bp and contains six exons interrupted by five introns. The gene has a typical promoter containing the sequences TATAAA and CCAAT 28 and 52 bp upstream of the cap site, respectively. They found striking similarities in genomic organization as well as in the promoter sequences between the human and rat PAP genes. The human PAP gene was mapped to chromosome 2p12 using rodent-human hybrid cells and in situ chromosomal hybridization. This localization coincides with that of the reg/lithostathine gene, which encodes a pancreatic secretory protein structurally related to PAP, suggesting that both genes derived from the same ancestral gene by duplication. 35 refs., 4 figs., 1 tab.

  14. Molecular cloning, expression, purification and crystallographic analysis of PRRSV 3CL protease

    SciTech Connect

    Tian, Xinsheng; Feng, Youjun; Zhao, Tiezhu; Peng, Hao; Yan, Jinghua; Qi, Jianxun; Jiang, Fan; Tian, Kegong; Gao, Feng

    2007-08-01

    Recombinant PRRSV 3CL protease was crystallized and the crystals diffracted to 2.1 Å resolution. 3CL protease, a viral chymotrypsin-like proteolytic enzyme, plays a pivotal role in the transcription and replication machinery of many RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the full-length 3CL protease from PRRSV was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that diffracted to 2.1 Å resolution and belong to space group C2, with unit-cell parameters a = 112.31, b = 48.34, c = 42.88 Å, β = 109.83°. The Matthews coefficient and the solvent content were calculated to be 2.49 Å{sup 3} Da{sup −1} and 50.61%, respectively, for one molecule in the asymmetric unit.

  15. Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from bufo melanostictus.

    PubMed

    Liu, Wa; Ji, Senlin; Zhang, A-Mei; Han, Qinqin; Feng, Yue; Song, Yuzhu

    2013-01-01

    Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.

  16. Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis

    SciTech Connect

    Owttrim, G.W.; Coleman, J.R.

    1987-05-01

    A recA-like gene isolated from the cyan