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Sample records for agouti gene product

  1. Isolation and characterization of Agouti: a diabetes/obesity related gene

    DOEpatents

    Woychik, Richard P.

    2000-06-27

    The present invention relates to the cloning and expression of the Agouti gene and analogous genes in transformed, transfected and transgenic mice. The present invention provides an animal model for the study of diabetes, obesity and tumors for the testing of potential therapeutic agents. The present invention provides oligonucleotide probes for the detection of the Agouti gene and mutations in the gene. The present invention also relates to the isolation and recombinant production of the Agouti gene product, production of antibodies to the Agouti gene product and their use as diagnostic and therapeutic agents.

  2. Isolation and characterization of Agouti: a diabetes/obesity related gene

    DOEpatents

    Woychik, Richard P.

    1998-01-01

    The present invention relates to the cloning and expression of the Agouti gene and analogous genes in transformed, transfected and transgenic mice. The present invention provides an animal model for the study of diabetes, obesity and tumors for the testing of potential therapeutic agents. The present invention provides oligonucleotide probes for the detection of the Agouti gene and mutations in the gene. The present invention also relates to the isolation and recombinant production of the Agouti gene product, production of antibodies to the Agouti gene product and their use as diagnostic and therapeutic agents.

  3. Molecular analysis of the mouse agouti gene and the role of dominant agouti-locus mutations in obesity and insulin resistance

    SciTech Connect

    Klebig, M.L.; Woychik, R.P.; Wilkinson, J.E.

    1994-09-01

    The lethal yellow (A{sup y/-}) and viable yellow (A{sup vy/-}) mouse agouti mutants have a predominantly yellow pelage and display a complex syndrome that includes obesity, hyperinsulinemia, and insulin resistance, hallmark features of obesity-associated noninsulin-dependent diabetes mellitus (NIDDM) in humans. A new dominant agouti allele, A{sup iapy}, has recently been identified; like the A{sup vy} allele, it is homozygous viable and confers obesity and yellow fur in heterozygotes. The agouti gene was cloned and characterized at the molecular level. The gene is expressed in the skin during hair growth and is predicted to encode a 131 amino acid protein, that is likely to be a secreted factor. In both Ay/- and A{sup iapy}/- mice, the obesity and other dominant pleiotropic effects are associated with an ectopic expression of agouti in many tissues where the gene product is normally not produced. In Ay, a 170-kb deletion has occurred that causes an upstream promoter to drive the ectopic expression of the wild-type agouti coding exons. In A{sup iapy}, the coding region of the gene is expressed from a cryptic promoter within the LTR of an intracisternal A-particle (IAP), which has integrated within the region just upstream of the first agouti coding exon. Transgenic mice ubiquitously expressing the cloned agouti gene under the influence of the beta-actin and phosphoglycerate kinase promoters display obesity, hyperinsulinemia, and yellow coat color. This demonstrates unequivocally that ectopic expression of agouti is responsible for the yellow obese syndrome.

  4. Analysis of the function of the agouti gene in obesity and diabetes

    SciTech Connect

    Mynatt, R.L.; Miltenberger, R.J.; Klebig, M.L.

    1996-09-01

    This chapter discusses the agouti gene and dominant mutations in that gene that lead to agouti-induced obesity, and recent work with transgenic mice to elucidate the role of agouti in obesity. Agouti was cloned in 1992 by the lab of Rick Woychik at Oak Ridge National Laboratory, making it the first of many recently cloned mouse obesity genes. Sequence analysis predicted that mouse agouti is a secreted protein of 131 amino acids. The mature protein has a basic central region (lys57-arg85), a proline-rich domain (pro86-pro91) and a C-terminal region (cys 92-cys 13 1) containing 10 cysteine residues which form 5 disulfide bonds. The human homologue of agouti has also been cloned by the Woychik lab and maps to human chromosome 20q 11.2. Human agouti is 132 amino acids long and is 85% similar to the mouse agouti protein and is normally expressed in adipose tissue. The researchers have been able to recapitulate obesity, hyperinsulinemia, and hyperglycemia with the ubiquitous expression of agouti. Agouti expression in either liver and adipose tissue alone does not cause obesity, and there`s a dose-dependent effect of agouti on body weight, food efficiency, body temperature, and insulin and glucose levels.

  5. Molecular structure and chromosomal mapping of the human homolog of the agouti gene

    SciTech Connect

    Kwon, H.Y.; Woychik, R.P.; Bultman, S.J. |; Loeffler, C.; Hansmann, I.; Chen, W.J.; Furdon, P.J.; Wilkison, W.; Powell, J.G.; Usala, A.L.

    1994-10-11

    The agouti (a) locus in mouse chromosome 2 normally regulates coat color pigmentation. The mouse agouti gene was recently cloned and shown to encode a distinctive 131-amino acid protein with a consensus signal peptide. Here the authors describe the cloning of the human homolog of the mouse agouti gene using an interspecies DNA-hybridization approach. Sequence analysis revealed that the coding region of the human agouti gene is 85% identical to the mouse gene and has the potential to encode a protein of 132 amino acids with a consensus signal peptide. Chromosomal assignment using somatic-cell-hybrid mapping panels and fluorescence in situ hybridization demonstrated that the human agouti gene maps to chromosome band 20q11.2. This result revealed that the human agouti gene is closely linked to several traits, including a locus called MODY (for maturity onset diabetes of the young) and another region that is associated with the development of myeloid leukemia. Initial expression studies with RNA from several adult human tissues showed that the human agouti gene is expressed in adipose tissue and testis.

  6. The insertion of a full-length Bos taurus LINE element is responsible for a transcriptional deregulation of the Normande Agouti gene.

    PubMed

    Girardot, Michael; Guibert, Sylvain; Laforet, Marie-Pierre; Gallard, Yves; Larroque, Hélène; Oulmouden, Ahmad

    2006-08-01

    Mammalian pigmentation is controlled by the concerted action of Tyr, Tyrp1 and Dct producing eumelanin and/or pheomelanin in melanocytes. The ratio of these two pigments is determined by the agonist alpha-melanocyte stimulating hormone and the antagonist Agouti protein acting on the Mc1r. Here we show that the Agouti gene is over-expressed in Normande breed compared with Prim'Holstein breed. The Normande cattle have a characteristic coat color phenotype with a variable presence of black (eumelanin) hair over a red/brown background. We have found a previously undescribed full-length L1-BT element inserted in the 5'-genomic sequence of the Agouti gene in Normande cattle which promotes the over-expression of alternative transcripts. The variable expression of the alternative transcript directed by the long interspersed nuclear element promoter may be the origin of the brindle coat color pattern of the Normande breed. This new bovine Agouti allele isolated in Normande breed has been named Abr. Finally, as ectopic over-expression of Agouti in Ay mice is responsible for the obesity syndrome, we discuss the possible consequences of Abr for meat and milk production in cattle.

  7. Ectopic expression of the agouti gene in transgenic mice causes obesity, features of type II diabetes, and yellow fur.

    PubMed

    Klebig, M L; Wilkinson, J E; Geisler, J G; Woychik, R P

    1995-05-23

    Mice that carry the lethal yellow (Ay) or viable yellow (Avy) mutation, two dominant mutations of the agouti (a) gene in mouse chromosome 2, exhibit a phenotype that includes yellow fur, marked obesity, a form of type II diabetes associated with insulin resistance, and an increased susceptibility to tumor development. Molecular analyses of these and several other dominant "obese yellow" a-locus mutations suggested that ectopic expression of the normal agouti protein gives rise to this complex pleiotropic phenotype. We have now tested this hypothesis directly by generating transgenic mice that ectopically express an agouti cDNA clone encoding the normal agouti protein in all tissues examined. Transgenic mice of both sexes have yellow fur, become obese, and develop hyperinsulinemia. In addition, male transgenic mice develop hyperglycemia by 12-20 weeks of age. These results demonstrate conclusively that the ectopic agouti expression is responsible for most, if not all, of the phenotypic traits of the dominant, obese yellow mutants.

  8. Characterization of the dog agouti gene and a nonagouti mutation in german shepherd dogs

    SciTech Connect

    Kerns, Julie A.; Newton, J.; Berryere, Tom G.; Rubin, Edward M.; Cheng, Jan-Fang; Schmutz, Sheila M.; Barsh, Gregory S.

    2004-07-08

    The interaction between two genes, Agouti and Melanocortin-1 receptor (Mc1r), produces diverse pigment patterns in mammals by regulating the type, amount, and distribution pattern of the two pigment types found in mammalian hair: eumelanin (brown/black) and pheomelanin (yellow/red). In domestic dogs (Canis familiaris), there is a tremendous variation in coat color patterns between and within breeds; however, previous studies suggest that the molecular genetics of pigment-type switching in dogs may differ from that of other mammals. Here we report the identification and characterization of the Agouti gene from domestic dogs, predicted to encode a 131-amino-acid secreted protein 98 percent identical to the fox homolog, and which maps to chromosome CFA24 in a region of conserved linkage. Comparative analysis of the Doberman Pinscher Agouti cDNA, the fox cDNA, and 180 kb of Doberman Pinscher genomic DNA suggests that, as with laboratory mice, different pigment-type-switching patterns in the canine family are controlled by alternative usage of different promoters and untranslated first exons. A small survey of Labrador Retrievers, Greyhounds, Australian Shepherds, and German Shepherd Dogs did not uncover any polymorphisms, but we identified a single nucleotide variant in black German Shepherd Dogs predicted to cause an Arg-to-Cys substitution at codon 96, which is likely to account for recessive inheritance of a uniform black coat.

  9. Combined effects of insulin treatment and adipose tissue-specific agouti expression on the development of obesity.

    PubMed

    Mynatt, R L; Miltenberger, R J; Klebig, M L; Zemel, M B; Wilkinson, J E; Wilkinson, W O; Woychik, R P

    1997-02-04

    The agouti gene product is a secreted protein that acts in a paracrine manner to regulate coat color in mammals. Several dominant mutations at the agouti locus in mice cause the ectopic, ubiquitous expression of agouti, resulting in a condition similar to adult-onset obesity and non-insulin-dependent diabetes mellitus. The human agouti protein is 85% homologous to mouse agouti; however, unlike the mouse agouti gene, human agouti is normally expressed in adipose tissue. To address whether expression of agouti in human adipose tissue is physiologically relevant, transgenic mice were generated that express agouti in adipose tissue. Similar to most humans, these mice do not become obese or diabetic. However, we found that daily insulin injections significantly increased weight gain in the transgenic lines expressing agouti in adipose tissue, but not in nontransgenic mice. These results suggest that insulin triggers the onset of obesity and that agouti expression in adipose tissue potentiates this effect. Accordingly, the investigation of agouti's role in obesity and non-insulin-dependent diabetes mellitus in mice holds significant promise for understanding the pathophysiology of human obesity.

  10. Ectopic expression of the agouti gene in transgenic mice causes obesity, features of type II diabetes, and yellow fur

    SciTech Connect

    Klebig, M.L.; Woychik, R.P.; Wilkinson, J.E.; Geisler, J.G. |

    1995-05-23

    Mice that carry the lethal yellow (A{sup y}) or viable yellow (A{sup vy}) mutation, two dominant mutations of the agouti (a) gene in mouse chromosome 2, exhibit a phenotype that includes yellow fur, marked obesity, a form of type II diabetes associated with insulin resistance, and an increased susceptibility to tumor development. Molecular analyses of these and several other dominant {open_quotes}obese yellow{close_quotes} a-locus mutations suggested that ectopic expression of the normal agouti protein gives rise to this complex pleiotropic phenotype. We have now tested this hypothesis directly by generating transgenic mice that ectopically express an agouti cDNA clone encoding the normal agouti protein in all tissues examined. Transgenic mice of both sexes have yellow fur, become obese, and develop hyperinsulinemia. In addition, male transgenic mice develop hyperglycemia by 12-20 weeks of age. These results demonstrate conclusively that the ectopic agouti expression is responsible for most, if not all, of the phenotypic traits of the dominant, obese yellow mutants. 42 refs., 5 figs.

  11. Agouti signalling protein (ASIP) gene: molecular cloning, sequence characterisation and tissue distribution in domestic goose.

    PubMed

    Zhang, J; Wang, C; Liu, Y; Liu, J; Wang, H Y; Liu, A F; He, D Q

    2016-06-01

    Agouti signalling protein (ASIP) is an endogenous antagonist of melanocortin-1 receptor (MC1R) and is involved in the regulation of pigmentation in mammals. The objective of this study was to identify and characterise the ASIP gene in domestic goose. The goose ASIP cDNA consisted of a 44-nucleotide 5'-terminal untranslated region (UTR), a 390-nucleotide open-reading frame (ORF) and a 45-nucleotide 3'-UTR. The length of goose ASIP genomic DNA was 6176 bp, including three coding exons and two introns. Bioinformatic analysis indicated that the ORF encodes a protein of 130 amino-acid residues with a molecular weight of 14.88 kDa and an isoelectric point of 9.73. Multiple sequence alignments and phylogenetic analysis showed that the amino-acid sequence of ASIP was conserved in vertebrates, especially in the avian species. RT-qPCR showed that the goose ASIP mRNA was differentially expressed in the pigment deposition tissues, including eye, foot, feather follicle, skin of the back, as well as in skin of the abdomen. The expression level of the ASIP gene in skin of the abdomen was higher than that in skin of the back. Those findings will contribute to further understanding the functions of the ASIP gene in geese plumage colouring.

  12. Liver-specific expression of the agouti gene in transgenic mice promotes liver carcinogenesis in the absence of obesity and diabetes

    SciTech Connect

    Kuklin, Alexander; Mynatt, Randall; Klebig, Mitch; Kiefer, Laura; Wilkison, William O; Woychik, Richard P; Michaud III, Edward J

    2004-01-01

    Background: The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals. Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1 receptor (Mc1r) on melanocytes. Lethal yellow (Ay) and viable yellow (Avy) are dominant regulatory mutations in the mouse agouti gene that cause the wild- ype protein to be produced at abnormally high levels throughout the body. Mice harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia, hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver. The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed by its ability to inhibit binding of the -melanocyte stimulating hormone ( MSH) to the Mc1r. Body weight, plasma insulin and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a single intraperitoneal injection (10 mg/kg) of the hepatocellular carcinogen, diethylnitrosamine (DEN), at 15 days of age. Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights were recorded. Results: The albumin-agouti transgene was expressed at high levels in the livers of mice and produced a functional agouti protein. Albumin-agouti transgenic mice had normal body weights and normal levels of blood glucose and plasma insulin, but responded to chemical initiation of the liver with an increased number

  13. Structural and Molecular Evolutionary Analysis of Agouti and Agouti-Related Proteins

    PubMed Central

    Jackson, Pilgrim J.; Douglas, Nick R.; Chai, Biaoxin; Binkley, Jonathan; Sidow, Arend; Barsh, Gregory S.; Millhauser, Glenn L.

    2010-01-01

    Summary Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products. PMID:17185225

  14. Agouti regulation of intracellular calcium: Role in the insulin resistance of viable yellow mice

    SciTech Connect

    Zemel, M.B.; Kim, J.H.; Woychik, R.P.; Michaud, E.J.; Hadwell, S.H.; Patel, I.R.; Wilkison, W.O.

    1995-05-23

    Several dominant mutations at the agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the agouti gene product mediates these effects is unclear. Since intracellular Ca{sup 2+} is believed to play a role in mediating insulin action and dysregulation of Ca{sup 2+} flux is observed in diabetic animals and humans, we examined the status of intracellular Ca{sup 2+} in mice carrying the dominant agouti allele, viable yellow (A{sup vy}). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca{sup 2+}]{sub i}) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the agouti gene product is capable of inducing increased [Ca{sup 2+}]{sub i} in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca{sup 2+}]{sub i}. 36 refs., 3 figs., 2 tabs.

  15. Molecular characterization of the mouse agouti locus.

    PubMed

    Bultman, S J; Michaud, E J; Woychik, R P

    1992-12-24

    The agouti (a) locus acts within the microenvironment of the hair follicle to regulate coat color pigmentation in the mouse. We have characterized a gene encoding a novel 131 amino acid protein that we propose is the one gene associated with the agouti locus. This gene is normally expressed in a manner consistent with a locus function, and, more importantly, its structure and expression are affected by a number of representative alleles in the agouti dominance hierarchy. In addition, we found that the pleiotropic effects associated with the lethal yellow (Ay) mutation, which include pronounced obesity, diabetes, and the development of neoplasms, are accompanied by deregulated overexpression of the agouti gene in numerous tissues of the adult animal.

  16. Epistatic Interaction of the Melanocortin 1 Receptor and Agouti Signaling Protein Genes Modulates Wool Color in the Brazilian Creole Sheep.

    PubMed

    Hepp, Diego; Gonçalves, Gislene Lopes; Moreira, Gilson Rudinei Pires; de Freitas, Thales Renato Ochotorena

    2016-11-01

    Different pigmentation genes have been associated with color diversity in domestic animal species. The melanocortin 1 receptor (MC1R), agouti signaling protein (ASIP), tyrosinase-related protein 1 (TYRP1), and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) genes are candidate genes responsible for variation in wool color among breeds of sheep. Although the influence of these genes has been described in some breeds, in many others the effect of interactions among genes underlying wool color has not been investigated. The Brazilian Creole sheep is a local breed with a wide variety of wool color, ranging from black to white with several intermediate hues. We analyzed in this study the influence of the genes MC1R, ASIP, TYRP1, and KIT on the control of wool color in this breed. A total of 410 samples were analyzed, including 148 white and 262 colored individuals. The MC1R and ASIP polymorphisms were significantly associated with the segregation of either white or colored wool. The dominant MC1R allele (E(D) p.M73K and p.D121N) was present only in colored animals. All white individuals were homozygous for the MC1R recessive allele (E(+)) and carriers of the duplicated copy of ASIP A gene expression assay showed that only the carrier of the duplicated copy of ASIP produces increased levels in skin, not detectable in the single homozygous copy. These results demonstrate that the epistatic interaction of the genotypes in the MC1R and ASIP gene is responsible for the striking color variation in the Creole breed.

  17. Coat colours in the Massese sheep breed are associated with mutations in the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes.

    PubMed

    Fontanesi, L; Dall'Olio, S; Beretti, F; Portolano, B; Russo, V

    2011-01-01

    Massese is an Italian dairy sheep breed characterized by animals with black skin and horns and black or apparent grey hairs. Owing to the presence of these two coat colour types, this breed can be considered an interesting model to evaluate the effects of coat colour gene polymorphisms on this phenotypic trait. Two main loci have been already shown to affect coat colour in sheep: Agouti and Extension coding for the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes, respectively. The Agouti locus is affected by a large duplication including the ASIP gene that may determine the Agouti white and tan allele (A(Wt)). Other disrupting or partially inactivating mutations have been identified in exon 2 (a deletion of 5 bp, D(5); and a deletion of 9 bp, D(9)) and in exon 4 (g.5172T>A, p.C126S) of the ASIP gene. Three missense mutations in the sheep MC1R gene cause the dominant black E(D) allele (p.M73K and p.D121N) and the putative recessive e allele (p.R67C). Here, we analysed these ASIP and MC1R mutations in 161 Massese sheep collected from four flocks. The presence of one duplicated copy allele including the ASIP gene was associated with grey coat colour (P = 9.4E-30). Almost all animals with a duplicated copy allele (37 out of 41) showed uniform apparent grey hair and almost all animals without a duplicated allele (117 out of 120) were completely black. Different forms of duplicated alleles were identified in Massese sheep including, in almost all cases, copies with exon 2 disrupting or partially inactivating mutations making these alleles different from the A(Wt) allele. A few exceptions were observed in the association between ASIP polymorphisms and coat colour: three grey sheep did not carry any duplicated copy allele and four black animals carried a duplicated copy allele. Of the latter four sheep, two carried the E(D) allele of the MC1R gene that may be the cause of their black coat colour. The coat colour of all other black animals may be

  18. Polymorphism of the goat agouti signaling protein gene and its relationship with coat color in Italian and Spanish breeds.

    PubMed

    Badaoui, B; D'Andrea, M; Pilla, F; Capote, J; Zidi, A; Jordana, J; Ferrando, A; Delgado, J V; Martínez, A; Vidal, O; Amills, M

    2011-08-01

    Agouti signaling protein (ASIP) is one of the key players in the modulation of hair pigmentation in mammals. Binding to the melanocortin 1 receptor, ASIP induces the synthesis of phaeomelanin, associated with reddish brown, red, tan, and yellow coats. We have sequenced 2.8 kb of the goat ASIP gene in 48 individuals and identified two missense (Cys126Gly and Val128Gly) and two intronic polymorphisms. In silico analysis revealed that the Cys126Gly substitution may cause a structural change by disrupting a highly conserved disulfide bond. We studied its segregation in 12 Spanish and Italian goat breeds (N = 360) with different pigmentation patterns and found striking differences in the frequency of the putative loss-of-function Gly(126) allele (Italian 0.43, Spanish Peninsular 0.08), but we did not observe a clear association with coat color. This suggests that the frequency of this putative loss-of-function allele has evolved under the influence of demographic rather than selection factors in goats from these two geographical areas.

  19. Maternal epigenetics and methyl supplements affect agouti gene expression in A{sup vy}/a mice

    SciTech Connect

    Wolff, G.L.

    1998-08-01

    Viable yellow (A{sup vy}/a) mice are larger, obese, hyperinsulinemic, more susceptible to cancer, and, on average, shorter lived than their non-yellow siblings. They are epigenetic mosaics ranging from a yellow phenotype with maximum ectopic agouti overexpression, through a continuum of mottled agouti/yellow phenotypes with partial agouti overexpression, to a pseudoagouti phenotype with minimal ectopic expression. Pseudoagouti A{sup vy}/a mice are lean, healthy, and longer lived than their yellow siblings. Here the authors report that feeding pregnant black a/a dams methyl-supplemented diets alters epigenetic regulation of agouti expression in their offspring, as indicated by increased agouti/black mottling in the direction of the pseudoagouti phenotype. They also present confirmatory evidence that epigenetic phenotypes are maternally heritable. Thus A{sup vy} expression, already known to be modulated by imprinting, strain-specific modification, and maternal epigenetic inheritance, is also modulated by maternal diet. These observations suggest, at least in this special case, that maternal dietary supplementation may positively affect health and longevity of the offspring. Therefore, this experimental system should be useful for identifying maternal factors that modulate epigenetic mechanisms, especially DNA methylation, in developing embryos.

  20. Copy number variation and missense mutations of the agouti signaling protein (ASIP) gene in goat breeds with different coat colors.

    PubMed

    Fontanesi, L; Beretti, F; Riggio, V; Gómez González, E; Dall'Olio, S; Davoli, R; Russo, V; Portolano, B

    2009-01-01

    In goats, classical genetic studies reported a large number of alleles at the Agouti locus with effects on coat color and pattern distribution. From these early studies, the dominant A(Wt) (white/tan) allele was suggested to cause the white color of the Saanen breed. Here, we sequenced the coding region of the goat ASIP gene in 6 goat breeds (Girgentana, Maltese, Derivata di Siria, Murciano-Granadina, Camosciata delle Alpi, and Saanen), with different coat colors and patterns. Five single nucleotide polymorphisms (SNPs) were identified, 3 of which caused missense mutations in conserved positions of the cysteine-rich carboxy-terminal domain of the protein (p.Ala96Gly, p.Cys126Gly, and p.Val128Gly). Allele and genotype frequencies suggested that these mutations are not associated or not completely associated with coat color in the investigated goat breeds. Moreover, genotyping and sequencing results, deviation from Hardy-Weinberg equilibrium, as well as allele copy number evaluation from semiquantitative fluorescent multiplex PCR, indicated the presence of copy number variation (CNV) in all investigated breeds. To confirm the presence of CNV and evaluate its extension, we applied a bovine-goat cross-species array comparative genome hybridization (aCGH) experiment using a custom tiling array based on bovine chromosome 13. aCGH results obtained for 8 goat DNA samples confirmed the presence of CNV affecting a region of less that 100 kb including the ASIP and AHCY genes. In Girgentana and Saanen breeds, this CNV might cause the A(Wt) allele, as already suggested for a similar structural mutation in sheep affecting the ASIP and AHCY genes, providing evidence for a recurrent interspecies CNV. However, other mechanisms may also be involved in determining coat color in these 2 breeds.

  1. Disruption of the RIIβ subunit of PKA reverses the obesity syndrome of agouti lethal yellow mice

    PubMed Central

    Czyzyk, Traci A.; Sikorski, Maria A.; Yang, Linghai; McKnight, G. Stanley

    2008-01-01

    Agouti lethal yellow (Ay) mice express agouti ectopically because of a genetic rearrangement at the agouti locus. The agouti peptide is a potent antagonist of the melanocortin 4 receptor (MC4R) expressed in neurons, and this leads to hyperphagia, hypoactivity, and increased fat mass. The MC4R signals through Gs and is thought to stimulate the production of cAMP and activation of downstream cAMP effector molecules such as PKA. Disruption of the RIIβ regulatory subunit gene of PKA results in release of the active catalytic subunit and an increase in basal PKA activity in cells where RIIβ is highly expressed. Because RIIβ is expressed in neurons including those in the hypothalamic nuclei where MC4R is prominent we tested the possibility that the RIIβ knockout might rescue the body weight phenotypes of the Ay mice. Disruption of the RIIβ PKA regulatory subunit gene in mice leads to a 50% reduction in white adipose tissue and resistance to diet-induced obesity and hyperglycemia. The RIIβ mutation rescued the elevated body weight, hyperphagia, and obesity of Ay mice. Partial rescue of the Ay phenotypes was even observed on an RIIβ heterozygote background. These results suggest that the RIIβ gene mutation alters adiposity and locomotor activity by modifying PKA signaling pathways downstream of the agouti antagonism of MC4R in the hypothalamus. PMID:18172198

  2. Agouti regulates adipocyte transcription factors.

    PubMed

    Mynatt, R L; Stephens, J M

    2001-04-01

    Agouti is a secreted paracrine factor that regulates pigmentation in hair follicle melanocytes. Several dominant mutations cause ectopic expression of agouti, resulting in a phenotype characterized by yellow fur, adult-onset obesity and diabetes, increased linear growth and skeletal mass, and increased susceptibility to tumors. Humans also produce agouti protein, but the highest levels of agouti in humans are found in adipose tissue. To mimic the human agouti expression pattern in mice, transgenic mice (aP2-agouti) that express agouti in adipose tissue were generated. The transgenic mice develop a mild form of obesity, and they are sensitized to the action of insulin. We correlated the levels of specific regulators of insulin signaling and adipocyte differentiation with these phenotypic changes in adipose tissue. Signal transducers and activators of transcription (STAT)1, STAT3, and peroxisome proliferator-activated receptor (PPAR)-gamma protein levels were elevated in the transgenic mice. Treatment of mature 3T3-L1 adipocytes recapitulated these effects. These data demonstrate that agouti has potent effects on adipose tissue. We hypothesize that agouti increases adiposity and promotes insulin sensitivity by acting directly on adipocytes via PPAR-gamma.

  3. Agouti revisited: transcript quantification of the ASIP gene in bovine tissues related to protein expression and localization.

    PubMed

    Albrecht, Elke; Komolka, Katrin; Kuzinski, Judith; Maak, Steffen

    2012-01-01

    Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.

  4. Agouti Revisited: Transcript Quantification of the ASIP Gene in Bovine Tissues Related to Protein Expression and Localization

    PubMed Central

    Albrecht, Elke; Komolka, Katrin; Kuzinski, Judith; Maak, Steffen

    2012-01-01

    Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species. PMID:22530003

  5. A combination of probiotics and whey proteins enhances anti-obesity effects of calcium and dairy products during nutritional energy restriction in aP2-agouti transgenic mice.

    PubMed

    Yoda, Kazutoyo; Sun, Xiaocum; Kawase, Manabu; Kubota, Akira; Miyazawa, Kenji; Harata, Gaku; Hosoda, Masataka; Hiramatsu, Masaru; He, Fang; Zemel, Michael B

    2015-06-14

    Lactobacillus rhamnosus GG, Lactobacillus paracasei TMC0409, Streptococcus thermophilus TMC1543 and whey proteins were used to prepare fermented milk. For the experiment aP2- agouti transgenic mice were pre-treated with a high-sucrose/high-fat diet for 6 weeks to induce obesity. The obese mice were fed a diet containing 1·2% Ca and either non-fat dried milk (NFDM) or probiotic-fermented milk (PFM) with nutritional energy restriction for 6 weeks. The animals were examined after the treatment for changes in body weight, fat pad weight, fatty acid synthase (FAS) activity, lypolysis, the expression levels of genes related to lipid metabolism, insulin sensitivity in adipocytes and skeletal muscle and the presence of biomarkers for oxidative and inflammatory stress in plasma. It was found that the PFM diet significantly reduced body weight, fat accumulation, and adipocyte FAS activity, and increased adipocyte lipolysis as compared with the effects of the NFDM diet (P<0·05). The adipose tissue gene expression of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) was significantly suppressed in mice that were fed PFM as compared with those that were fed NFDM (P<0·05). PFM caused a greater up-regulation of skeletal muscle PPARα, PPARδ, uncoupling protein 3 (UCP3) and GLUT4 expression and a significant decrease in the plasma concentration of insulin, malondialdehyde, TNF-α, monocyte chemotactic protein-1 and C-reactive protein as compared with the effects of NFDM (P<0·05). Fermentation of milk with selected probiotics and supplementation of milk with whey proteins may thus enhance anti-obesity effects of Ca and dairy products by the suppression of adipose tissue lipogenesis, activation of fat oxidation in skeletal muscle and reduction of oxidative and inflammatory stress.

  6. Gene expression analysis indicates CB1 receptor upregulation in the hippocampus and neurotoxic effects in the frontal cortex 3 weeks after single-dose MDMA administration in Dark Agouti rats

    PubMed Central

    2013-01-01

    Background 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") is a widely used recreational drug known to impair cognitive functions on the long-run. Both hippocampal and frontal cortical regions have well established roles in behavior, memory formation and other cognitive tasks and damage of these regions is associated with altered behavior and cognitive functions, impairments frequently described in heavy MDMA users. The aim of this study was to examine the hippocampus, frontal cortex and dorsal raphe of Dark Agouti rats with gene expression arrays (Illumina RatRef bead arrays) looking for possible mechanisms and new candidates contributing to the effects of a single dose of MDMA (15 mg/kg) 3 weeks earlier. Results The number of differentially expressed genes in the hippocampus, frontal cortex and the dorsal raphe were 481, 155, and 15, respectively. Gene set enrichment analysis of the microarray data revealed reduced expression of 'memory’ and 'cognition’, 'dendrite development’ and 'regulation of synaptic plasticity’ gene sets in the hippocampus, parallel to the upregulation of the CB1 cannabinoid- and Epha4, Epha5, Epha6 ephrin receptors. Downregulated gene sets in the frontal cortex were related to protein synthesis, chromatin organization, transmembrane transport processes, while 'dendrite development’, 'regulation of synaptic plasticity’ and 'positive regulation of synapse assembly’ gene sets were upregulated. Changes in the dorsal raphe region were mild and in most cases not significant. Conclusion The present data raise the possibility of new synapse formation/synaptic reorganization in the frontal cortex three weeks after a single neurotoxic dose of MDMA. In contrast, a prolonged depression of new neurite formation in the hippocampus is suggested by the data, which underlines the particular vulnerability of this brain region after the drug treatment. Finally, our results also suggest the substantial contribution of CB1 receptor and

  7. Molecular basis of the pleiotropic phenotype of mice carrying the hypervariable yellow (A{sup hvy}) mutation at the agouti locus

    SciTech Connect

    Argeson, A.C.; Nelson, K.K.; Siracusa, L.D.

    1996-02-01

    The murine agouti locus regulates a switch in pigment synthesis between eumelanin (black/brown pigment) and phaeomelanin (yellow/red pigment) by hair bulb melanocytes. We recently described a spontaneous mutation, hypervariable yellow (A{sup hvy}) and demonstrated that A{sup hvy} is responsible for the largest range of phenotypes yet identified at the agouti locus, producing mice that are obese with yellow coats to mice that are of normal weight with black coats. Here, we show that agouti expression is altered both temporally and spatially in A{sup hvy} mutants. Agouti expression levels are positively correlated with the degree of yellow pigmentation in individual A{sup hvy} mice, consistent with results from other dominant yellow agouti mutations. Sequencing of 5{prime} RACE and genomic PCR products revealed that A{sup hvy} resulted from the integration of an intracisternal A particle (IAP) in an antisense orientation within the 5{prime} untranslated agouti exon 1C. This retrovirus-like element is responsible for deregulating agouti expression in A{sup hvy} mice; agouti expression is correlated with the methylation state of CpG residues in the IAP long terminal repeat as well as in host genomic DNA. In addition, the data suggest that the variable phenotype of A{sup hvy} offspring is influenced in part by the phenotype of their A{sup hvy} female parent. 42 refs., 7 figs., 1 tab.

  8. Peptoid mimics of agouti related protein.

    PubMed

    Thompson, Darren A; Chai, Biao-Xin; Rood, Hilary L E; Siani, Michael A; Douglas, Nicholai R; Gantz, Ira; Millhauser, Glenn L

    2003-04-17

    The Agouti Related Protein (AGRP) is an endogenous antagonist of melanocortin-3 and -4 receptors, each of which plays a key role in body weight homeostasis. We designed a peptoid trimer based on AGRP 111-113 in which a single chiral atom is used to partially restrain the backbone structure. Peptoid 5 displaced both radiolabeled Nle4-alpha-MSH (IC(50)=3.1 microM) and AGRP (86-132) (IC(50)=1.9 microM) from the human melanocortin-4 receptor and functioned as an antagonist of alpha-MSH stimulated cAMP generation, thus providing an important lead in the development of AGRP mimetics.

  9. Characterization, tissue distribution and regulation by fasting of the agouti family of peptides in the sea bass (Dicentrarchus labrax).

    PubMed

    Agulleiro, Maria Josep; Cortés, Raúl; Leal, Esther; Ríos, Diana; Sánchez, Elisa; Cerdá-Reverter, José Miguel

    2014-09-01

    The melanocortin system is one of the most complex hormonal systems in vertebrates. Atypically, the signaling of melanocortin receptors is regulated by the binding of endogenous antagonists, named agouti-signaling protein (ASIP) and agouti-related protein (AGRP). Teleost specific genome duplication (TSGD) rendered new gene copies in teleost fish and up to four different genes of the agouti family of peptides have been characterized. In this paper, molecular cloning was used to characterize mRNA of the agouti family of peptides in sea bass. Four different genes were identified: AGRP1, ASIP1, AGRP2 and ASIP2. The AGRP1 gene is mainly expressed in the brain whereas ASIP1 is mainly expressed in the ventral skin. Both ASIP2 and AGRP2 are expressed in the brain and the pineal gland but also in some peripheral tissues. Immunocytochemical studies demonstrated that AGRP1 is exclusively expressed within the lateral tuberal nucleus, the homologue of the mammalian arcuate nucleus in fish. Long-term fasting (8-29 days) increased the hypothalamic expression of AGRP1 but depressed AGRP2 expression (15-29 days). In contrast, the hypothalamic expression of ASIP2 was upregulated during short-term fasting suggesting that this peptide could be involved in the short term regulation of food intake in the sea bass.

  10. Placentation in the capybara (Hydrochaerus hydrochaeris), Agouti (Dasyprocta aguti) and paca (Agouti paca).

    PubMed

    Miglino, M A; Carter, A M; dos Santos Ferraz, R H; Fernandes Machado, M R

    2002-05-01

    Placentae of three hystricimorph rodents--capybara, agouti and paca--were examined by conventional histology, immunohistochemistry for cytokeratin and vimentin, and TUNEL staining. The placentae were divided into lobules of labyrinthine syncytium separated by interlobular and marginal trophoblast. The subplacenta comprised cytotrophoblasts, supported on lamellae of allantoic mesoderm, and syncytiotrophoblast. The central excavation was still apparent in the definitive placenta of capybara. In agouti and paca, the decidua of the junctional zone formed a mesoplacenta comprising a capsule and a pedicle. Towards term the pedicle formed a tenuous attachment between placenta and uterine wall comprising a few maternal vessels surrounded by degraded tissue. In paca placenta, it was shown by TUNEL staining that breakdown of this tissue occurred by apoptosis. The visceral yolk sac was highly villous and, in agouti, the yolk sac villi were extremely long. Lateral to its attachment to the placenta, the fetal surface was covered with non-vascular yolk sac endoderm. A layer of spongiotrophoblast cells was interposed between the endoderm and the marginal trophoblast.

  11. The early origin of melanocortin receptors, agouti-related peptide, agouti signalling peptide, and melanocortin receptor-accessory proteins, with emphasis on pufferfishes, elephant shark, lampreys, and amphioxus.

    PubMed

    Västermark, Ake; Schiöth, Helgi B

    2011-06-11

    There are conflicting theories about the evolution of melanocortin MC receptors while only few studies have addressed the evolution of agouti-related peptide (AgRP) and agouti signalling peptide (ASIP), which are antagonists at the melanocortin receptors (MCRs), or the melanocortin MC(2) receptor accessory proteins (MRAP1 and MRAP2). Previously we have cloned melanocortin MC receptors (MC(a) and MC(b)) genes in river lamprey and here we identify orthologues to these melanocortin MC receptor sequences in the sea lamprey. We investigate the putative presence of the melanocortin MC receptor genes in lancelet (amphioxus; Branchiostoma floridae) but we find it unlikely that such gene exists, due to a sharp drop in sequence similarity beyond sequence clusters of known receptors. We show the presence of AgRP and ASIP in elephant shark, a cartilaginous fish belonging to the subclass of Elasmobranchii. However, we do not find any of these genes in lamprey or lancelet after detailed analysis of both targeted and whole proteome regular expression scans. We found MRAP2, but not MRAP1, to be present in elephant shark and sea lamprey while Fugu (T. rubripes) has both genes. This study shows that the most ancient presence of these melanocortin-related sequences is found in elephant shark and lampreys considering the current available sequence data.

  12. Agouti expression in human adipose tissue: functional consequences and increased expression in type 2 diabetes.

    PubMed

    Smith, Steven R; Gawronska-Kozak, Barbara; Janderová, Lenka; Nguyen, Taylor; Murrell, Angela; Stephens, Jacqueline M; Mynatt, Randall L

    2003-12-01

    It is well recognized that the agouti/melanocortin system is an important regulator of body weight homeostasis. Given that agouti is expressed in human adipose tissue and that the ectopic expression of agouti in adipose tissue results in moderately obese mice, the link between agouti expression in human adipose tissue and obesity/type 2 diabetes was investigated. Although there was no apparent relationship between agouti mRNA levels and BMI, agouti mRNA levels were significantly elevated in subjects with type 2 diabetes. The regulation of agouti in cultured human adipocytes revealed that insulin did not regulate agouti mRNA, whereas dexamethasone treatment potently increased the levels of agouti mRNA. Experiments with cultured human preadipocytes and with cells obtained from transgenic mice that overexpress agouti demonstrated that melanocortin receptor (MCR) signaling in adipose tissue can regulate both preadipocyte proliferation and differentiation. Taken together, these results reveal that agouti can regulate adipogenesis at several levels and suggest that there are functional consequences of elevated agouti levels in human adipose tissue. The influence of MCR signaling on adipogenesis combined with the well-established role of MCR signaling in the hypothalamus suggest that adipogenesis is coordinately regulated with food intake and energy expenditure.

  13. Deletion of eIF2beta suppresses testicular cancer incidence and causes recessive lethality in agouti-yellow mice.

    PubMed

    Heaney, Jason D; Michelson, Megan V; Youngren, Kirsten K; Lam, Man-Yee J; Nadeau, Joseph H

    2009-04-15

    The agouti-yellow (A(y)) deletion is the only genetic modifier known to suppress testicular germ cell tumor (TGCT) susceptibility in mice or humans. The A(y) mutation deletes Raly and Eif2s2, and induces the ectopic expression of agouti, all of which are potential TGCT-modifying mutations. Here we report that the reduced TGCT incidence of heterozygous A(y) males and the recessive embryonic lethality of A(y) are caused by the deletion of Eif2s2, the beta subunit of translation initiation factor eIF2. We found that the incidence of affected males was reduced 2-fold in mice that were partially deficient for Eif2s2 and that embryonic lethality occurred near the time of implantation in mice that were fully deficient for Eif2s2. In contrast, neither reduced expression of Raly in gene-trap mice nor ectopic expression of agouti in transgenic or viable-yellow (A(vy)) mutants affected TGCT incidence or embryonic viability. In addition, we provide evidence that partial deficiency of Eif2s2 attenuated germ cell proliferation and differentiation, both of which are important to TGCT formation. These results show that germ cell development and TGCT pathogenesis are sensitive to the availability of the eIF2 translation initiation complex and to changes in the rate of translation.

  14. Functional anatomy of the female genital organs of the wild black agouti (Dasyprocta fuliginosa) female in the Peruvian Amazon.

    PubMed

    Mayor, P; Bodmer, R E; Lopez-Bejar, M

    2011-02-01

    This study examined anatomical and histological characteristics of genital organs of 38 black agouti females in the wild in different reproductive stages, collected by rural hunters in the North-eastern Peruvian Amazon. Females in the follicular phase of the estrous cycle had greater antral follicle sizes than other females, the largest antral follicle measuring 2.34mm. Antral follicles in pregnant females and females in luteal phase of the estrous cycle had an average maximum diameter smaller than 1mm. In black agouti females in follicular phase, some antral follicles are selected to continue to growth, reaching a pre-ovulatory diameter of 2mm. Mean ovulation rate was 2.5 follicles and litter size was 2.1 embryos or fetuses per pregnant female, resulting in a rate of ovum mortality of 20.8%. Many follicles from which ovulation did not occur of 1-mm maximum diameter luteinize forming accessory CL. The constituent active luteal tissues of the ovary are functional and accessory CL. Although all females had accessory CL, transformation of follicles into accessory CL occurred especially in pregnant females, resulting in a contribution from 9% to 23% of the total luteal volume as pregnancy advances. The persistence of functional CL throughout pregnancy might reflect the importance for the maintenance of gestation and may be essential for the continuous hormonal production. The duplex uterus of the agouti female is composed by two completely independent uterine horns with correspondent separate cervices opening into the vagina. In pregnant females, most remarkable observed uterine adaptations were induced by the progressive enlargement caused by the normal pregnancy evolution. The wild black agouti showed different vaginal epithelium features in accordance with the reproductive state of the female.

  15. Molecular characterization of a region of DNA associated with mutations at the agouti locus in the mouse.

    PubMed

    Bultman, S J; Russell, L B; Gutierrez-Espeleta, G A; Woychik, R P

    1991-09-15

    Molecular characterization of a radiation-induced agouti (a)-locus mutation has resulted in the isolation of a segment of DNA that maps at or near the a locus on chromosome 2 in the mouse. This region of DNA is deleted in several radiation- or chemical-induced homozygous-lethal a-locus mutations and is associated with specific DNA structural alterations in two viable a-locus mutations. We propose that DNA probes from this region of chromosome 2 will be useful for ultimately characterizing the individual gene or genes associated with a-locus function.

  16. Agouti signaling protein stimulates cell division in "viable yellow" (A vy/a) mouse liver

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enhanced linear growth, hyperplasia, and tumorigenesis are well-known characteristics of "viable yellow" agouti Avy/- mice (1); however, the functional basis for this aspect of the phenotype is unknown. In the present study, we ascertained whether agouti signaling protein (ASIP) levels in Avy/a or a...

  17. Leptin responsiveness in mice that ectopically express agouti protein.

    PubMed

    Harris, Ruth B S; Mitchell, Tiffany D; Mynatt, Randall L

    Agouti protein is an endogenous antagonist of melanocortin receptors (MCR), including MCR3 and MCR4, which have been implicated as part of the hypothalamic mechanism that mediates leptin-induced hypophagia. In this experiment we examined the effects of peripheral and central leptin administration in male and female beta-actin promoter (BAPa) mice that express agouti protein ectopically and have a phenotype that includes obesity and diabetes which is exaggerated in males compared with females. Intraperitoneal infusion of 10 microg leptin/day for 13 days caused weight loss and a transient inhibition of food intake in wild-type mice, with a greater effect in males than females. Male BAPa mice were resistant to leptin infusion whereas female mice lost weight. All of the mice lost body weight following a single intracerebroventricular injection of leptin but the effect was greater in female BAPa mice than any other group. There also was a delayed suppression of food intake that was the same for wild-type and BAPa female mice, whereas food intake recovered faster in BAPa than wild-type males. The dissociation between food intake and body weight loss implies a significant effect of leptin on energy expenditure in BAPa mice. These results demonstrate that the effect of leptin on energy balance is not entirely dependent upon the melanocortin system.

  18. Regulation of PPARgamma and obesity by agouti/melanocortin signaling in adipocytes.

    PubMed

    Mynatt, Randall L; Stephens, Jacquelins M

    2003-06-01

    To study the potential biological role of agouti/melanocortin signaling in human adipose tissue, we engineered transgenic mice to overexpress agouti in adipose tissue. The aP2-agouti transgenic mice become significantly heavier than littermates. The increased body weight is maintained at approximately 15% above nontransgenic mice through 20 weeks and is caused by increased fat mass. The obesity is increased by a high-fat diet. There is no change in food intake in the aP2-agouti mice suggesting changes in energy utilization. A possible mechanism is that the agouti/melanocortin signaling regulates levels of PPARgamma. PPARgamma functions as a major regulator of adipocyte differentiation and as a receptor for the antidiabetic thiazolidinediones. Agouti increases PPARgamma protein levels in differentiated 3T3-L1 adipocytes, and PPARgamma expression is elevated in the fat pads of the aP2-agouti transgenic mice. The modest weight gain observed in the transgenic mice suggests that hypothalamic pathways regulating food intake are intact and the observed adiposity is within ranges that can be achieved by a paracrine mechanism at the adipocyte level.

  19. Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice

    SciTech Connect

    Engle, S.J.; Chen, J.; Tischfield, J.A.

    1994-09-01

    Adenine phosphoribosyltransferase (APRT: EC 2.4.2.7), a ubiquitously expressed purine salvage enzyme, catalyzes the synthesis of AMP and inorganic pyrophosphate from existing adenine and 5-phosphoribosyl-1-pyrophosphate. Deficiency of this enzyme in humans results in the accumulation of 2,8-dihydroxyadenine leading to crystalluria and nephrolithiasis. In order to facilitate our study of this rare, autosomal recessive disorder, we applied the advances in gene targeting technology and mouse embryonic stem (ES) cell culture to the production of APRT-deficient mice. A positive-negative targeting strategy was used. The tageting vector contain 5.6 kb of the mouse APRT gene, a neomycin resistance gene in exon 3 as a positive selection marker, and a HSV thymidine kinase gene at the 3{prime} end of the homology as a negative selection marker. The vector was introduced into D3 ES cells by electroporation and the cells were selected for G418 and ganciclovir (GANC) resistance. G418-GANC resistant clones were screened by Southern blot. One of several correctly targeted clones was expanded and used for blastocyst microinjection to produce chimeric mice. Chimeric animals were bred and agouti progeny heterozygous for the targeted allele were obtained. Heterozygous animals have been bred to produce APRT-deficient animals. Matings are currently underway to determine the phenotype of APRT/HPRT-deficient animals.

  20. Diet-induced hypermethylation at agouti viable yellow is not inherited transgenerationally through the female

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of nonmutagenic environmental exposures can sometimes be transmitted for several generations, suggesting transgenerational inheritance of induced epigenetic variation. Methyl donor supplementation of female mice during pregnancy induces CpG hypermethylation at the agouti viable yellow (A...

  1. Who's behind that mask and cape? The Asian leopard cat's Agouti (ASIP) allele likely affects coat colour phenotype in the Bengal cat breed.

    PubMed

    Gershony, L C; Penedo, M C T; Davis, B W; Murphy, W J; Helps, C R; Lyons, L A

    2014-12-01

    Coat colours and patterns are highly variable in cats and are determined mainly by several genes with Mendelian inheritance. A 2-bp deletion in agouti signalling protein (ASIP) is associated with melanism in domestic cats. Bengal cats are hybrids between domestic cats and Asian leopard cats (Prionailurus bengalensis), and the charcoal coat colouration/pattern in Bengals presents as a possible incomplete melanism. The complete coding region of ASIP was directly sequenced in Asian leopard, domestic and Bengal cats. Twenty-seven variants were identified between domestic and leopard cats and were investigated in Bengals and Savannahs, a hybrid with servals (Leptailurus serval). The leopard cat ASIP haplotype was distinguished from domestic cat by four synonymous and four non-synonymous exonic SNPs, as well as 19 intronic variants, including a 42-bp deletion in intron 4. Fifty-six of 64 reported charcoal cats were compound heterozygotes at ASIP, with leopard cat agouti (A(P) (be) ) and domestic cat non-agouti (a) haplotypes. Twenty-four Bengals had an additional unique haplotype (A2) for exon 2 that was not identified in leopard cats, servals or jungle cats (Felis chaus). The compound heterozygote state suggests the leopard cat allele, in combination with the recessive non-agouti allele, influences Bengal markings, producing a darker, yet not completely melanistic coat. This is the first validation of a leopard cat allele segregating in the Bengal breed and likely affecting their overall pelage phenotype. Genetic testing services need to be aware of the possible segregation of wild felid alleles in all assays performed on hybrid cats.

  2. Identification of genes and gene products necessary for bacterial bioluminescence.

    PubMed

    Engebrecht, J; Silverman, M

    1984-07-01

    Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymatic activities necessary for light production. We report the results of a genetic analysis to identify the luminescence genes (lux) that reside on this recombinant plasmid. lux gene mutations were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by complementation in trans with a series of polar transposon insertions on other plasmids. lux genes were defined by complementation of lux gene defects on pairs of plasmids in trans in E. coli. Hybrid plasmids were also used to direct the synthesis of polypeptides in the E. coli minicell system. Seven lux genes and the corresponding gene products were identified from the complementation analysis and the minicell programing experiments. These genes, in the order of their position on a linear map, and the apparent molecular weights of the gene products are luxR (27,000), luxI (25,000), luxC (53,000), luxD (33,000), luxA (40,000), luxB (38,000), and luxE (42,000). From the luminescence phenotypes of E. coli containing mutant plasmids, functions were assigned to these genes: luxA, luxB, luxC, luxD, and luxE encode enzymes for light production and luxR and luxI encode regulatory functions.

  3. [Ovarian activity of Agouti paca (Rodentia: Agoutidae) under captivity].

    PubMed

    Montes Pérez, Rubén C; Cabrera Baz, Elsy A

    2006-09-01

    The ovarian activity of Agouti paca was characterized by hormonal profiles and ovarian structures. Samples of blood were taken from eight females (seven adults and one juvenile) at the breeding grounds of the Facultad de Medicina Veterinaria y Zootecnia in Yucatśn, Mexico. Sampling lasted approximately two months and was done every three and six days. Blood was collected from anesthetized animals, and the levels of progesterone (P4) and 17 beta estradiol (E2) were analized by radioimmunoassay technique. Macroscopic and microscopic analyses were carried out in ovaries of dead animals. The estrous cycle lasted 29+/-8.4 days, levels of 1.61+/-0.65 ng/ml for P4 and 39+/-24 pg/ml for E2 were observed for a follicular phase, 6.18+/-3.70 ng/ml and 29+/-16 pg/ml for P4 and E2 respectively in the luteal phase. Statistically significant differences were found between phases for P4 but not for E2. The presence of extragonadal steroids with levels of P4 of 1.9+/-0.77 ng/ml and E2 of 22+/-17 pg/ml were observed, which are not produced by the effects of managing stress. The changes in the levels of P4 during the cycle are indicators of luteal activity, with the intersticial tissue acting probably as active steroids-producing gland. Follicular growth was observed during the entire cycle.

  4. Agouti sequence polymorphisms in coyotes, wolves and dogs suggest hybridization.

    PubMed

    Schmutz, Sheila M; Berryere, Thomas G; Barta, Jodi L; Reddick, Kimberley D; Schmutz, Josef K

    2007-01-01

    Domestic dogs have been shown to have multiple alleles of the Agouti Signal Peptide (ASIP) in exon 4 and we wished to determine the level of polymorphism in the common wild canids of Canada, wolves and coyotes, in comparison. All Canadian coyotes and most wolves have banded hairs. The ASIP coding sequence of the wolf did not vary from the domestic dog but one variant was detected in exon 4 of coyotes that did not alter the arginine at this position. Two other differences were found in the sequence flanking exon 4 of coyotes compared with the 45 dogs and 1 wolf. The coyotes also demonstrated a relatively common polymorphism in the 3' UTR sequence that could be used for population studies. One of the ASIP alleles (R96C) in domestic dogs causes a solid black coat color in homozygotes. Although some wolves are melanistic, this phenotype does not appear to be caused by this same mutation. However, one wolf, potentially a dog-wolf hybrid or descendant thereof, was heterozygous for this allele. Likewise 2 coyotes, potentially dog-coyote or wolf-coyote hybrid descendants, were heterozygous for the several polymorphisms in and flanking exon 4. We could conclude that these were coyote-dog hybrids because both were heterozygous for 2 mutations causing fawn coat color in dogs.

  5. Human AZU-1 gene, variants thereof and expressed gene products

    DOEpatents

    Chen, Huei-Mei; Bissell, Mina

    2004-06-22

    A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

  6. Design, pharmacology, and NMR structure of a minimized cystine knot with agouti-related protein activity.

    PubMed

    Jackson, Pilgrim J; McNulty, Joseph C; Yang, Ying-Kui; Thompson, Darren A; Chai, Biaoxin; Gantz, Ira; Barsh, Gregory S; Millhauser, Glenn L

    2002-06-18

    The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The NMR structure of this active domain was recently determined and suggested that melanocortin receptor contacts were made primarily by two loops presented by a well-structured cystine knot domain within AGRP(87-132) [McNulty et al. (2001) Biochemistry 40, 15520-15527]. This hypothesis is tested here with NMR structure and activity studies of a 34-residue AGRP analogue designed to contain only the cystine knot domain. The designed miniprotein folds to a homogeneous product, retains the desired cystine knot architecture, functions as an antagonist, and maintains the melanocortin receptor pharmacological profile of AGRP(87-132). The AGRP-like activity of this molecule supports the hypothesis that indeed the cystine knot region possesses the melanocortin receptor contact points. Moreover, this potent AGRP analogue is synthetically accessible, may serve in the development of therapeutics for the treatment of diseases related to energy balance. and may also find use as a new reagent for probing melanocortin receptor structure and function.

  7. [Immune response genes products in human physiology].

    PubMed

    Khaitov, R M; Alekseev, L P

    2012-09-01

    Current data on physiological role of human immune response genes' proteomic products (antigens) are discussed. The antigens are specified by a very high level of diversity that mediates a wide specter ofphysiological functions. They actually provide integrity and biological stability of human as species. These data reveal new ideas on many pathological processes as well as drafts new approaches for prophylaxis and treatment.

  8. Transient ectopic overexpression of agouti-signalling protein 1 (asip1) induces pigment anomalies in flatfish.

    PubMed

    Guillot, Raúl; Ceinos, Rosa Maria; Cal, Rosa; Rotllant, Josep; Cerdá-Reverter, José Miguel

    2012-01-01

    While flatfish in the wild exhibit a pronounced countershading of the dorso-ventral pigment pattern, malpigmentation is commonly observed in reared animals. In fish, the dorso-ventral pigment polarity is achieved because a melanization inhibition factor (MIF) inhibits melanoblast differentiation and encourages iridophore proliferation in the ventrum. A previous work of our group suggested that asip1 is the uncharacterized MIF concerned. In order to further support this hypothesis, we have characterized asip1 mRNAs in both turbot and sole and used deduced peptide alignments to analyze the evolutionary history of the agouti-family of peptides. The putative asip precursors have the characteristics of a secreted protein, displaying a putative hydrophobic signal. Processing of the potential signal peptide produces mature proteins that include an N-terminal region, a basic central domain with a high proportion of lysine residues as well as a proline-rich region that immediately precedes the C-terminal poly-cysteine domain. The expression of asip1 mRNA in the ventral area was significantly higher than in the dorsal region. Similarly, the expression of asip1 within the unpigmented patches in the dorsal skin of pseudoalbino fish was higher than in the pigmented dorsal regions but similar to those levels observed in the ventral skin. In addition, the injection/electroporation of asip1 capped mRNA in both species induced long term dorsal skin paling, suggesting the inhibition of the melanogenic pathways. The data suggest that fish asip1 is involved in the dorsal-ventral pigment patterning in adult fish, where it induces the regulatory asymmetry involved in precursor differentiation into mature chromatophore. Adult dorsal pseudoalbinism seems to be the consequence of the expression of normal developmental pathways in an inaccurate position that results in unbalanced asip1 production levels. This, in turn, generates a ventral-like differentiation environment in dorsal regions.

  9. [The influence of hyperleptinemia during pregnancy on fetal weight and obesity development in progeny mice with agouti yellow mutation].

    PubMed

    Makarova, E N; Syracheva, M S; Bazhan, N M

    2014-03-01

    Maternal obesity increases the risk of obesity in the offspring, and obesity is accompanied by an increase in blood leptin levels. Leptin can influence the progeny metabolism via its influence on fetal growth and, possibly, via its action on AgRP expression in placenta. The "yellow" mutation at the mouse agouti locus (A(y)) evokes obesity and increases blood leptin levels in pregnant mice. The aim was to examine the influence of A(y) mutation in pregnant mice on fetal weight, placental expression of AgRP gene and food intake and obesity development in progeny. A(y) pregnant females as compared to control ones had increased circulating leptin levels on days 13 and 18 of pregnancy. Both fetal weight and placental expression of AgRP gene were increased on day 13 of pregnancy and decreased on day 18 of pregnancy in A(y) females as compared to control ones. Both control (a/a) and obesity prone (A(y)/a) male young born to A(y) mothers had lowered body weight and enhanced food intake between 5 and 11 weeks of age as compared to male progeny of control mothers. The enhanced leptin levels during pregnancy in mice are associated with retardation of obesity development in obesity prone male offspring and with changes in fetal weight and AgRP gene expression in placenta.

  10. Interactions between straw size and thawing rates on the cryopreservation of agouti (Dasyprocta aguti) epididymal sperm.

    PubMed

    Silva, M A; Peixoto, G C X; Sousa, P C; Bezerra, F S B; Bezerra, A C D S; Silva, A R

    2012-02-01

    This study verifies the interactions between straw size and thawing rates and their impact on the epididymal sperm from this species. Caudae epididymidum from 10 agoutis were subjected to retrograde washing using a coconut water extender (ACP-109c(®) ). Epididymal sperm were evaluated and extended in ACP-109c(®) plus egg yolk (20%) and glycerol (6%). The samples were packaged in 0.25- or 0.50-ml straws, frozen in liquid nitrogen and thawed at 37°C/1 min or 70°C/8 s, followed by a re-evaluation. The use of 0.25-ml straws thawed at 37°C/1 min provided a value of 26.6% for sperm motility. No interactions between straw size and thawing rates were verified on agouti sperm (p > 0.05), but when 0.5-ml straws were thawed at 70°C/8 s, sperm vigour decreased significantly (p < 0.05). It is recommended that the agouti epididymal sperm cryopreserved in ACP-109c(®) extender should be packaged in 0.25- or 0.50-ml straws and thawed at 37°C/60 s.

  11. Hypothalamic Agouti-Related Peptide mRNA is Elevated During Natural and Stress-Induced Anorexia.

    PubMed

    Dunn, I C; Wilson, P W; D'Eath, R B; Boswell, T

    2015-09-01

    As part of their natural lives, animals can undergo periods of voluntarily reduced food intake and body weight (i.e. animal anorexias) that are beneficial for survival or breeding, such as during territorial behaviour, hibernation, migration and incubation of eggs. For incubation, a change in the defended level of body weight or 'sliding set point' appears to be involved, although the neural mechanisms reponsible for this are unknown. We investigated how neuropeptide gene expression in the arcuate nucleus of the domestic chicken responded to a 60-70% voluntary reduction in food intake measured both after incubation and after an environmental stressor involving transfer to unfamiliar housing. We hypothesised that gene expression would not change in these circumstances because the reduced food intake and body weight represented a defended level in birds with free access to food. Unexpectedly, we observed increased gene expression of the orexigenic peptide agouti-related peptide (AgRP) in both incubating and transferred animals compared to controls. Also pro-opiomelanocortin (POMC) mRNA was higher in incubating hens and significantly increased 6 days after exposure to the stressor. Conversely expression of neuropeptide Y and cocaine- and amphetamine-regulated transcript gene was unchanged in both experimental situations. We conclude that AgRP expression remains sensitive to the level of energy stores during natural anorexias, which is of adaptive advantage, although its normal orexigenic effects are over-ridden by inhibitory signals. In the case of stress-induced anorexia, increased POMC may contribute to this inhibitory role, whereas, for incubation, reduced feeding may also be associated with increased expression in the hypothalamus of the anorexigenic peptide vasoactive intestinal peptide.

  12. Regulation of photoreceptor gene transcription via a highly conserved transcriptional regulatory element by vsx gene products

    PubMed Central

    Pan, Yi; Comiskey, Daniel F.; Kelly, Lisa E.; Chandler, Dawn S.

    2016-01-01

    Purpose The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site. Methods X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos. Results We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities. Conclusions vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system. PMID:28003732

  13. Structural and ultrastructural features of the agouti tongue (Dasyprocta aguti Linnaeus, 1766).

    PubMed

    Ciena, Adriano Polican; de Sousa Bolina, Cristina; de Almeida, Sonia Regina Yokomizo; Rici, Rose Eli Grassi; de Oliveira, Moacir Franco; da Silva, Marcelo Cavenaghi Pereira; Miglino, Maria Angélica; Watanabe, Ii-sei

    2013-08-01

    The agouti (Dasyprocta aguti Linnaeus, 1766) is a wild rodent belonging to the family Dasyproctidae that is found throughout Brazil and feeds on fruits and seeds. The aim of the present study was to describe the following features of the tongue of agouti: its morphological structures, the three-dimensional characteristics of the lingual papillae surface, the connective tissue cores (CTCs) and the epithelial cell ultrastructure. Four types of papillae were observed on the dorsal surface of the tongue with a triangular shape: filiform, fungiform, foliate and vallate. Filiform papillae were distributed throughout the tongue surface, and removal of the epithelial surface revealed conical CTCs and multifilaments. Fungiform papillae were observed in the rostral and middle regions, whereas foliate papillae developed in pairs on the lateral margin of the caudal region. Removal of the epithelium in these regions revealed CTCs with parallel laminar conformation. Vallate papillae were arranged in a V-shape in the caudal region, and their CTCs ranged in shape from elongate to ovoid. The ultrastructural components of the dorsal epithelium were the basal, spinous, granular and keratinised layers. A broad area with cytoplasmic projections was identified in the interface region between the lamina propria and the basal layer. Flattened cells with intermediate filaments were observed in the transitional region between spinous and granular layers. The keratinised layer was composed of superimposed epithelial cells where desmosomes and cell-surface microridges were observed. These structural features, including the three-dimensional aspects of the lingual papillae, the CTCs and the epithelial ultrastructure, indicate that when compared with other animals, particularly other rodent species, the morphological features of the tongue of agouti are relatively well developed, especially regarding foliate and vallate papillae.

  14. Structural and ultrastructural features of the agouti tongue (Dasyprocta aguti Linnaeus, 1766)

    PubMed Central

    Ciena, Adriano Polican; Bolina, Cristina de Sousa; de Almeida, Sonia Regina Yokomizo; Rici, Rose Eli Grassi; de Oliveira, Moacir Franco; da da Silva, Marcelo Cavenaghi Pereira; Miglino, Maria Angélica; Watanabe, Ii-sei

    2013-01-01

    The agouti (Dasyprocta aguti Linnaeus, 1766) is a wild rodent belonging to the family Dasyproctidae that is found throughout Brazil and feeds on fruits and seeds. The aim of the present study was to describe the following features of the tongue of agouti: its morphological structures, the three-dimensional characteristics of the lingual papillae surface, the connective tissue cores (CTCs) and the epithelial cell ultrastructure. Four types of papillae were observed on the dorsal surface of the tongue with a triangular shape: filiform, fungiform, foliate and vallate. Filiform papillae were distributed throughout the tongue surface, and removal of the epithelial surface revealed conical CTCs and multifilaments. Fungiform papillae were observed in the rostral and middle regions, whereas foliate papillae developed in pairs on the lateral margin of the caudal region. Removal of the epithelium in these regions revealed CTCs with parallel laminar conformation. Vallate papillae were arranged in a V-shape in the caudal region, and their CTCs ranged in shape from elongate to ovoid. The ultrastructural components of the dorsal epithelium were the basal, spinous, granular and keratinised layers. A broad area with cytoplasmic projections was identified in the interface region between the lamina propria and the basal layer. Flattened cells with intermediate filaments were observed in the transitional region between spinous and granular layers. The keratinised layer was composed of superimposed epithelial cells where desmosomes and cell-surface microridges were observed. These structural features, including the three-dimensional aspects of the lingual papillae, the CTCs and the epithelial ultrastructure, indicate that when compared with other animals, particularly other rodent species, the morphological features of the tongue of agouti are relatively well developed, especially regarding foliate and vallate papillae. PMID:23701183

  15. Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina).

    PubMed

    Castelo, T S; Silva, A M; Bezerra, L G P; Costa, C Y M; Lago, A E A; Bezerra, J A B; Campos, L B; Praxedes, E C G; Silva, A R

    2015-12-01

    We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes-epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide - DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing-thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.

  16. An obesity-dependent lactation defect in the viable yellow agouti mouse is associated with mammary inflammation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maternal obesity is known to delay lactogenesis in breast-feeding women, as well as negatively impact lactation in other species. Obesity is also understood to be associated with inflammation. Work with the viable yellow agouti (Avy) mouse in our laboratory has documented a lactation defect in obese...

  17. Correlating information contents of gene ontology terms to infer semantic similarity of gene products.

    PubMed

    Gan, Mingxin

    2014-01-01

    Successful applications of the gene ontology to the inference of functional relationships between gene products in recent years have raised the need for computational methods to automatically calculate semantic similarity between gene products based on semantic similarity of gene ontology terms. Nevertheless, existing methods, though having been widely used in a variety of applications, may significantly overestimate semantic similarity between genes that are actually not functionally related, thereby yielding misleading results in applications. To overcome this limitation, we propose to represent a gene product as a vector that is composed of information contents of gene ontology terms annotated for the gene product, and we suggest calculating similarity between two gene products as the relatedness of their corresponding vectors using three measures: Pearson's correlation coefficient, cosine similarity, and the Jaccard index. We focus on the biological process domain of the gene ontology and annotations of yeast proteins to study the effectiveness of the proposed measures. Results show that semantic similarity scores calculated using the proposed measures are more consistent with known biological knowledge than those derived using a list of existing methods, suggesting the effectiveness of our method in characterizing functional relationships between gene products.

  18. Integrating Ontological Knowledge and Textual Evidence in Estimating Gene and Gene Product Similarity

    SciTech Connect

    Sanfilippo, Antonio P.; Posse, Christian; Gopalan, Banu; Tratz, Stephen C.; Gregory, Michelle L.

    2006-06-08

    With the rising influence of the Gene On-tology, new approaches have emerged where the similarity between genes or gene products is obtained by comparing Gene Ontology code annotations associ-ated with them. So far, these approaches have solely relied on the knowledge en-coded in the Gene Ontology and the gene annotations associated with the Gene On-tology database. The goal of this paper is to demonstrate that improvements to these approaches can be obtained by integrating textual evidence extracted from relevant biomedical literature.

  19. Determination and validation of principal gene products

    PubMed Central

    Tress, Michael L.; Wesselink, Jan-Jaap; Frankish, Adam; López, Gonzalo; Goldman, Nick; Löytynoja, Ari; Massingham, Tim; Pardi, Fabio; Whelan, Simon; Harrow, Jennifer; Valencia, Alfonso

    2009-01-01

    Motivation Alternative splicing has the potential to generate a wide range of protein isoforms. For many computational applications and for experimental research, it is important to be able to concentrate on the isoform that retains the core biological function. For many genes this is far from clear. Results We have combined five methods into a pipeline that allows us to detect the principal variant for a gene. Most of the methods were based on conservation between species, at the level of both gene and protein. The five methods used were the conservation of exonic structure, the detection of non-neutral evolution, the conservation of functional residues, the existence of a known protein structure and the abundance of vertebrate orthologues. The pipeline was able to determine a principal isoform for 83% of a set of well-annotated genes with multiple variants. PMID:18006548

  20. Inducible product gene expression technology tailored to bioprocess engineering.

    PubMed

    Weber, Wilfried; Fussenegger, Martin

    2007-10-01

    Bioprocess engineering has developed as a discipline to design optimal culture conditions and bioreactor operation protocols for production cell lines engineered for constitutive expression of desired protein pharmaceuticals. With the advent of heterologous gene regulation systems it has become possible to fine-tune expression of difficult-to-produce protein pharmaceuticals to optimal levels and to conditionally engineer cell metabolism for the best production performance. However, most of the small-molecules used to trigger expression of product or metabolic engineering product genes are incompatible with downstream processing regulations or process economics. Recent progress in product gene control design has resulted in the development of bioprocess-compatible regulation systems, which are responsive to physical parameters such as temperature or physiologic trigger molecules that are either an inherent part of host cell metabolism or intrinsic components of licensed protein-free cell culture media, such as redox status, vitamin H and gaseous acetaldehyde. While all of these systems have been shown to fine-tune product gene expression independent of the host cell metabolism some of them can be plugged into metabolic networks to capture critical physiologic parameters and convert them into an optimal production response. Assembly of individual product gene control modalities into synthetic networks has recently enabled construction of autonomously regulated time-delay or cell density-sensitive gene circuits, which trigger population-wide induction of product gene expression at a predefined time or culture density. We provide a comprehensive overview on the latest developments in the design of bioprocess-compatible product gene control systems.

  1. Functions of the gene products of Escherichia coli.

    PubMed Central

    Riley, M

    1993-01-01

    A list of currently identified gene products of Escherichia coli is given, together with a bibliography that provides pointers to the literature on each gene product. A scheme to categorize cellular functions is used to classify the gene products of E. coli so far identified. A count shows that the numbers of genes concerned with small-molecule metabolism are on the same order as the numbers concerned with macromolecule biosynthesis and degradation. One large category is the category of tRNAs and their synthetases. Another is the category of transport elements. The categories of cell structure and cellular processes other than metabolism are smaller. Other subjects discussed are the occurrence in the E. coli genome of redundant pairs and groups of genes of identical or closely similar function, as well as variation in the degree of density of genetic information in different parts of the genome. PMID:7508076

  2. Dark Agouti rat model of chemotherapy-induced mucositis: establishment and current state of the art.

    PubMed

    Vanhoecke, Barbara; Bateman, Emma; Mayo, Bronwen; Vanlancker, Eline; Stringer, Andrea; Thorpe, Daniel; Keefe, Dorothy

    2015-06-01

    Mucositis is a major oncological problem. The entire gastrointestinal and genitourinary tract and also other mucosal surfaces can be affected in recipients of radiotherapy, and/or chemotherapy. Major progress has been made in recent years in understanding the mechanisms of oral and small intestinal mucositis, which appears to be more prominent than colonic damage. This progress is largely due to the development of representative laboratory animal models of mucositis. This review focuses on the development and establishment of the Dark Agouti rat mammary adenocarcinoma model by the Mucositis Research Group of the University of Adelaide over the past 20 years to characterize the mechanisms underlying methotrexate-, 5-fluorouracil-, and irinotecan-induced mucositis. It also aims to summarize the results from studies using different animal model systems to identify new molecular and cellular markers of mucositis.

  3. Dark Agouti rat model of chemotherapy-induced mucositis: Establishment and current state of the art

    PubMed Central

    Vanhoecke, Barbara; Bateman, Emma; Mayo, Bronwen; Vanlancker, Eline; Thorpe, Daniel; Keefe, Dorothy

    2015-01-01

    Mucositis is a major oncological problem. The entire gastrointestinal and genitourinary tract and also other mucosal surfaces can be affected in recipients of radiotherapy, and/or chemotherapy. Major progress has been made in recent years in understanding the mechanisms of oral and small intestinal mucositis, which appears to be more prominent than colonic damage. This progress is largely due to the development of representative laboratory animal models of mucositis. This review focuses on the development and establishment of the Dark Agouti rat mammary adenocarcinoma model by the Mucositis Research Group of the University of Adelaide over the past 20 years to characterize the mechanisms underlying methotrexate-, 5-fluorouracil-, and irinotecan-induced mucositis. It also aims to summarize the results from studies using different animal model systems to identify new molecular and cellular markers of mucositis. PMID:25966981

  4. Quinolizidine alkaloids in Ormosia arborea seeds inhibit predation but not hoarding by agoutis (Dasyprocta leporina).

    PubMed

    Guimarães, Paulo Roberto; José, Juliana; Galetti, Mauro; Trigo, José Roberto

    2003-05-01

    Quinolizidine alkaloids (QAs) are secondary compounds found in seeds of many species of plants, possibly protecting them against pathogens and seed predators. QAs were isolated from Ormosia arborea seeds and bioassayed against red-rumped agoutis (Dasyprocta leporina, Rodentia: Caviomorpha) to verify if they inhibit seed predation and food hoarding (seed dispersal). Three treatments were used: (1) seeds of O. arborea, (2) palatable seeds of Mimusops coriacea (Sapotaceae) treated with MeOH, and (3) seeds of M. coriacea treated with QAs dissolved in MeOH in similar concentration to that present in O. arborea. Palatable seeds were significantly more preyed upon than seeds treated with QAs and Ormosia seeds, but QAs did not influence hoarding behavior. QAs in O. arborea may have a strong effect in avoiding seed predation by rodents, without reducing dispersal.

  5. [Participation of Agouti related peptide in machanisms of wakefulness-sleep cycle regulation].

    PubMed

    Romanova, I V; Mikhrina, A L

    2013-01-01

    Agouti-related protein (AGRP) is expresses in hypothalamic neurons in human and animals. Immunohistochemical study in rats Wistar rats demonstrates significant changes AGRP optical density in the neurons of arcuate hypothalamic nucleus as well as in processes in the hypothalamus and nucleus accumbens after the 6 hours of sleep deprivation (increase) and after 2 hours of post-deprivative sleep (decrease). Comparison of these results with earlier obtained shows the opposite trend changes in AGRP optical density and speed limiting enzyme of dopamine synthesis-tyrosine hydroxylase in the hypothalamus and in striatonigral system. The increase of AGRP was accompanied by a decrease of tyrosine hydroxylase and the decrease of AGRP, apposite, it increases. The obtained data demonstrate the role ofAGRP as a modulator of the functional activity of the dopaminergic brain neurons. The problem of the relationship of various functions of organism (food behavior, sleep, stress) is discusses by their participation in the regulation of the same neurotransmitter systems.

  6. Detection in vivo of a new gene product (gene III) of cauliflower mosaic virus

    PubMed Central

    Xiong, C.; Lebeurier, G.; Hirth, L.

    1984-01-01

    Cauliflower mosaic virus DNA contains six major open reading frames (ORFs). As only the mRNA corresponding to the transcription of gene VI and its translation product have been isolated, the identification in infected plants of products corresponding to the five other putative genes remains to be established. The present paper reports the detection of an ORF III product by means of antibodies raised against an NH2-terminal synthetic peptide of 19 amino acids corresponding to a sequence predicted from the nucleotide sequence of ORF III. The detection of this gene product raises the question of the mechanism of its expression. Images PMID:16593524

  7. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5' UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  8. Regulation of Cell and Gene Therapy Medicinal Products in Taiwan.

    PubMed

    Lin, Yi-Chu; Wang, Po-Yu; Tsai, Shih-Chih; Lin, Chien-Liang; Tai, Hsuen-Yung; Lo, Chi-Fang; Wu, Shiow-Ing; Chiang, Yu-Mei; Liu, Li-Ling

    2015-01-01

    Owing to the rapid and mature development of emerging biotechnology in the fields of cell culture, cell preservation, and recombinant DNA technology, more and more cell or gene medicinal therapy products have been approved for marketing, to treat serious diseases which have been challenging to treat with current medical practice or medicine. This chapter will briefly introduce the Taiwan Food and Drug Administration (TFDA) and elaborate regulation of cell and gene therapy medicinal products in Taiwan, including regulatory history evolution, current regulatory framework, application and review procedures, and relevant jurisdictional issues. Under the promise of quality, safety, and efficacy of medicinal products, it is expected the regulation and environment will be more flexible, streamlining the process of the marketing approval of new emerging cell or gene therapy medicinal products and providing diverse treatment options for physicians and patients.

  9. Gene analogue finder: a GRID solution for finding functionally analogous gene products

    PubMed Central

    Tulipano, Angelica; Donvito, Giacinto; Licciulli, Flavio; Maggi, Giorgio; Gisel, Andreas

    2007-01-01

    Background To date more than 2,1 million gene products from more than 100000 different species have been described specifying their function, the processes they are involved in and their cellular localization using a very well defined and structured vocabulary, the gene ontology (GO). Such vast, well defined knowledge opens the possibility of compare gene products at the level of functionality, finding gene products which have a similar function or are involved in similar biological processes without relying on the conventional sequence similarity approach. Comparisons within such a large space of knowledge are highly data and computing intensive. For this reason this project was based upon the use of the computational GRID, a technology offering large computing and storage resources. Results We have developed a tool, GENe AnaloGue FINdEr (ENGINE) that parallelizes the search process and distributes the calculation and data over the computational GRID, splitting the process into many sub-processes and joining the calculation and the data on the same machine and therefore completing the whole search in about 3 days instead of occupying one single machine for more than 5 CPU years. The results of the functional comparison contain potential functional analogues for more than 79000 gene products from the most important species. 46% of the analyzed gene products are well enough described for such an analysis to individuate functional analogues, such as well-known members of the same gene family, or gene products with similar functions which would never have been associated by standard methods. Conclusion ENGINE has produced a list of potential functionally analogous relations between gene products within and between species using, in place of the sequence, the gene description of the GO, thus demonstrating the potential of the GO. However, the current limiting factor is the quality of the associations of many gene products from non-model organisms that often have

  10. Pregnancy in Hystricomorpha: gestational age and embryonic-fetal development of agouti (Dasyprocta prymnolopha, Wagler 1831) estimated by ultrasonography.

    PubMed

    Sousa, F C A; Alves, F R; Fortes, E A M; Ferraz, M S; Machado Júnior, A A N; de Menezes, D J A; de Carvalho, M A M

    2012-10-01

    Thirty-one pregnant agoutis, between Days 9 and 103 of gestation (Day 1 = day of detection of sperm in the vaginal smear), underwent B-mode ultrasonography; gestational sac diameter (GSD), crown-rump length (CRL), embryonic-fetal diameter (EFD), and placenta diameter (PD) were measured. There were positive correlations (P < 0.05) between GSD and CRL (r = 0.98), GSD and PD (r = 0.88), CRL and PD (r = 0.86), days of gestation (DG) and CRL (r = 0.85), and DG and PD (r = 0.73). The gestational sac was first observed on Day 14. The embryo was first seen on Day 18 in 9/31 of pregnant agoutis and on Day 22 in 20/31 of pregnant agoutis. Heartbeats were detected from the Day 25 and placentas were observed in 100% of the animals from Day 25. Early limb bud and ossification of the fetal skull were identified on Days 27 (15/31) and 45 (24/31), respectively. Fetal orientation (head and body) was evident from Day 40, the stomach, liver and lungs were identified on Day 50, the kidneys were reliably seen only on Day 55, and the aorta and vena cava were seen on Day 70. The fetal bowel and the urinary bladder were the last structures to be observed (Day 85). Ultrasonography was effective for early pregnancy diagnosis in agouti and for obtaining information on embryonic and fetal structures that could be used to predict gestational age and birth, thereby contributing to their reproductive management in captivity.

  11. Recovery and cryopreservation of epididymal sperm from agouti (Dasiprocta aguti) using powdered coconut water (ACP-109c) and Tris extenders.

    PubMed

    Silva, M A; Peixoto, G C X; Santos, E A A; Castelo, T S; Oliveira, M F; Silva, A R

    2011-10-01

    The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE, Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability, membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk (20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37°C for 1 min, followed by re-evaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P < 0.05) after centrifugation and dilution. After sperm cryopreservation and thawing, there were (mean ± SEM) 26.5 ± 2.6% motile sperm with 2.6 ± 0.2 vigor in the ACP-109c (ACP Biotecnologia) group, which was significantly better than 9.7 ± 2.6% motile sperm with 1.2 ± 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly better extender for processing and cryopreserving these sperm.

  12. Molecular and genetic characterization of a radiation-induced structural rearrangement in mouse chromosome 2 causing mutations at the limb deformity and agouti loci.

    PubMed

    Woychik, R P; Generoso, W M; Russell, L B; Cain, K T; Cacheiro, N L; Bultman, S J; Selby, P B; Dickinson, M E; Hogan, B L; Rutledge, J C

    1990-04-01

    Molecular characterization of mutations in the mouse, particularly those involving agent-induced major structural alterations, is proving to be useful for correlating the structure and expression of individual genes with their function in the whole organism. Here we present the characterization of a radiation-induced mutation that simultaneously generated distinct alleles of both the limb deformity (ld) and agouti (a) loci, two developmentally important regions of chromosome 2 normally separated by 20 centimorgans. Cytogenetic analysis revealed that an interstitial segment of chromosome 17 (17B- 17C; or, possibly, 17A2-17B) had been translocated into the distal end of chromosome 2, resulting in a smaller-than-normal chromosome 17 (designated 17del) and a larger form of chromosome 2 (designated 2(17). Additionally, a large interstitial segment of the 2(17) chromosome, immediately adjacent and proximal to the insertion site, did not match bands 2E4-2H1 at corresponding positions on a normal chromosome 2. Molecular analysis detected a DNA rearrangement in which a portion of the ld locus was joined to sequences normally tightly linked to the a locus. This result, along with the genetic and cytogenetic data, suggests that the alleles of ld and a in this radiation-induced mutation, designated ldIn2 and ajIn2, were associated with DNA breaks caused by an inversion of an interstitial segment in the 2(17) chromosome.

  13. Defining MC1R regulation in human melanocytes by its agonist α-melanocortin and antagonists agouti signaling protein and β-defensin 3.

    PubMed

    Swope, Viki B; Jameson, Joshua A; McFarland, Kevin L; Supp, Dorothy M; Miller, William E; McGraw, Dennis W; Patel, Mira A; Nix, Matthew A; Millhauser, Glenn L; Babcock, George F; Abdel-Malek, Zalfa A

    2012-09-01

    The melanocortin 1 receptor (MC1R), a G(s) protein-coupled receptor, has an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human β-defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-melanocyte-stimulating hormone (α-MSH))-induced increase in the activities of adenylate cyclase and tyrosinase, the rate-limiting enzyme for melanogenesis. α-Melanocortin and forskolin, which activate adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C, increased, whereas exposure to UV radiation reduced, MC1R gene and membrane protein expression. Brief treatment with α-MSH resulted in MC1R desensitization, whereas continuous treatment up to 3 hours caused a steady rise in cAMP, suggesting receptor recycling. Pretreatment with agouti signaling protein or HBD3 prohibited responsiveness to α-MSH, but not forskolin, suggesting receptor desensitization by these antagonists. Melanocytes from different donors expressed different levels of the G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6, as well as β-arrestin 1. Therefore, in addition to the MC1R genotype, regulation of MC1R expression and activity is expected to affect human pigmentation and the responses to UV.

  14. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    PubMed

    Santoro, Raffaella; Lienemann, Philipp; Fussenegger, Martin

    2009-08-14

    Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.

  15. Natural Products Version 2.0: Connecting Genes to Molecules

    PubMed Central

    Walsh, Christopher T.; Fischbach, Michael A.

    2009-01-01

    Natural products have played a prominent role in the history of organic chemistry, and they continue to be important as drugs, biological probes, and targets of study for synthetic and analytical chemists. In this perspective, we explore how connecting Nature’s small molecules to the genes that encode them has sparked a renaissance in natural product research, focusing primarily on the biosynthesis of polyketides and nonribosomal peptides. We survey monomer biogenesis, coupling chemistries from templated and non-templated pathways, and the broad set of tailoring reactions and hybrid pathways that give rise to the diverse scaffolds and functionalization patterns of natural products. We conclude by considering two questions: What would it take to find all natural product scaffolds? What kind of scientists will be studying natural products in the future? PMID:20121095

  16. Id-1 gene and gene products as therapeutic targets for treatment of breast cancer and other types of carcinoma

    DOEpatents

    Desprez, Pierre-Yves; Campisi, Judith

    2014-08-19

    A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

  17. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

    PubMed

    Green, M R; Treisman, R; Maniatis, T

    1983-11-01

    When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

  18. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2017-03-21

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  19. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2016-03-01

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  20. Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products.

    PubMed Central

    Ronson, C W; Astwood, P M; Nixon, B T; Ausubel, F M

    1987-01-01

    We have sequenced two genes dctB and dctD required for the activation of the C4-dicarboxylate transport structural gene dctA in free-living Rhizobium leguminosarum. The hydropathic profile of the dctB gene product (DctB) suggested that its N-terminal region may be located in the periplasm and its C-terminal region in the cytoplasm. The C-terminal region of DctB was strongly conserved with similar regions of the products of several regulatory genes that may act as environmental sensors, including ntrB, envZ, virA, phoR, cpxA, and phoM. The N-terminal domains of the products of several regulatory genes thought to be transcriptional activators, including ntrC, ompR, virG, phoB and sfrA. In addition, the central and C-terminal regions of DctD were strongly conserved with the products of ntrC and nifA, transcriptional activators that require the alternate sigma factor rpoN (ntrA) as co-activator. The central region of DctD also contained a potential ATP-binding domain. These results are consistent with recent results that show that rpoN product is required for dctA activation, and suggest that DctB plus DctD-mediated transcriptional activation of dctA may be mechanistically similar to NtrB plus NtrC-mediated activation of glnA in E. coli. PMID:3671068

  1. Plasma agouti-related protein levels in women with anorexia nervosa.

    PubMed

    Moriya, Junko; Takimoto, Yoshiyuki; Yoshiuchi, Kazuhiro; Shimosawa, Tatsuo; Akabayashi, Akira

    2006-10-01

    Agouti-related protein (AGRP) is the competitive antagonist of alpha-melanocyte stimulating hormone (alpha-MSH) located at melanocortin receptors 3 and 4 (MC3R and MC4R), and also acts as an MC4R inverse agonist. Hypothalamic AGRP controls food intake and body weight in rodents. It has also been found in human plasma. To study the possibility of disturbances in melanocortin receptor-related peptides in eating disorders, plasma AGRP, alpha-MSH, and leptin levels were measured in 18 female patients with anorexia nervosa (AN) (age, 23.5+/-7.1 yr; body mass index (BMI) 14.5+/-1.8 kg/m(2)) and 17 age-matched female controls (age, 25.8+/-3.9 yr; BMI 20.2+/-1.6 kg/m(2)). Blood samples were collected after overnight fasting, and plasma peptides levels were measured using ELISA. Plasma AGRP levels increased significantly in AN patients when compared with controls (P<0.01) while plasma alpha-MSH levels were not significantly different. Plasma leptin levels decreased significantly in AN patients when compared with controls (P<0.001). In addition, plasma AGRP levels were negatively correlated with leptin (r=-0.41, P<0.01) and BMI (r=-0.40, P<0.05) in all subjects. In conclusion, plasma AGRP elevation may be related to energy homeostasis disturbance in AN, and in addition to leptin, peripheral AGRP levels could be used as a nutritional marker in AN patients.

  2. Agouti-related protein increases food hoarding more than food intake in Siberian hamsters.

    PubMed

    Day, Diane E; Bartness, Timothy J

    2004-01-01

    Agouti-related protein (AgRP), an endogenous melanocortin 3/4 receptor antagonist, appears to play an important role in the control of food intake and energy balance because exogenous administration in rats and overexpression in mice result in hyperphagia and body mass gain. Furthermore, arcuate nucleus AgRP mRNA is increased with fasting in laboratory rats and mice and is decreased with refeeding. In Siberian hamsters, fasting also increases arcuate nucleus AgRP mRNA, but these animals increase food hoarding, rather than food intake with refeeding. Therefore, we tested whether exogenous AgRP increased food hoarding in this species. Hamsters were trained in a hoarding/foraging apparatus to run a programmed number of wheel revolutions to earn food pellets. Four doses of AgRP-(83-132) or vehicle were injected into the third ventricle at the beginning of the dark phase, and food hoarding, food intake, and foraging were measured at various time points subsequently. Overall, food hoarding was stimulated as much as 10 times more than food intake, and both responses occurred as early as 1 h after injection. Food hoarding was increased the greatest at the lowest dose (0.1 nmol), whereas food intake was increased the greatest at the second lowest dose (1 nmol). Food intake and especially food hoarding were increased up to seven days after the AgRP injections. Foraging was increased at all AgRP doses except the highest dose (100 nmol). These results suggest that AgRP triggers the search for food in this species, and once they find it, hoarding predominates over eating.

  3. Characterization, tissue distribution, and regulation of agouti-related protein (AgRP), cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) in Atlantic salmon (Salmo salar).

    PubMed

    Murashita, Koji; Kurokawa, Tadahide; Ebbesson, Lars O E; Stefansson, Sigurd O; Rønnestad, Ivar

    2009-06-01

    Key peptide hormones involved in the control of appetite in vertebrates were identified, their genes characterized and their regulation studied in Atlantic salmon: two agouti-related proteins (AgRP), cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY). The AgRP-1 and AgRP-2 genes encode prepro-proteins of 142- and 117-amino acids, respectively. The deduced AgRP-2 protein has 10 cysteine residues in the C-terminal polycysteine domain, while the AgRP-1 lacks the 6th and 7th cysteine residues observed in other species. AgRP-1 was principally expressed in the pituitary and skin, while AgRP-2 was highly expressed in the mid-gut, red muscle and gonads. The CART gene, encoding 118-amino acids, was strongly expressed in the brain and eye. In addition to salmon CART, we identified three to six variants of the CART gene in lower vertebrates by mining available databases. The salmon NPY gene, encoding 100-amino acids, was mainly expressed in the brain and eye. AgRP-1 and CART mRNA levels in the brain decreased after 6 days of fasting while AgRP-2 and NPY showed no significant change, suggesting that AgRP-1 and CART are involved in feeding regulation in Atlantic salmon. The identification of multiple variants of these appetite-regulating genes emphasizes the importance to further investigate the complex regulation of these genes.

  4. Plasmid genes required for microcin B17 production.

    PubMed Central

    San Millán, J L; Kolter, R; Moreno, F

    1985-01-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production. PMID:2993228

  5. Natural product proteomining, a quantitative proteomics platform, allows rapid discovery of biosynthetic gene clusters for different classes of natural products.

    PubMed

    Gubbens, Jacob; Zhu, Hua; Girard, Geneviève; Song, Lijiang; Florea, Bogdan I; Aston, Philip; Ichinose, Koji; Filippov, Dmitri V; Choi, Young H; Overkleeft, Herman S; Challis, Gregory L; van Wezel, Gilles P

    2014-06-19

    Information on gene clusters for natural product biosynthesis is accumulating rapidly because of the current boom of available genome sequencing data. However, linking a natural product to a specific gene cluster remains challenging. Here, we present a widely applicable strategy for the identification of gene clusters for specific natural products, which we name natural product proteomining. The method is based on using fluctuating growth conditions that ensure differential biosynthesis of the bioactivity of interest. Subsequent combination of metabolomics and quantitative proteomics establishes correlations between abundance of natural products and concomitant changes in the protein pool, which allows identification of the relevant biosynthetic gene cluster. We used this approach to elucidate gene clusters for different natural products in Bacillus and Streptomyces, including a novel juglomycin-type antibiotic. Natural product proteomining does not require prior knowledge of the gene cluster or secondary metabolite and therefore represents a general strategy for identification of all types of gene clusters.

  6. Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and Agouti-related protein mRNA levels and body weight in rats.

    PubMed

    Kamegai, J; Tamura, H; Shimizu, T; Ishii, S; Sugihara, H; Wakabayashi, I

    2001-11-01

    Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic growth hormone secretagogues (GHSs), ghrelin specifically releases growth hormone (GH) after intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. Recently, we showed that a single central administration of ghrelin increased food intake and hypothalamic agouti-related protein (AGRP) gene expression in rodents, and the orexigenic effect of this peptide seems to be independent of its GH-releasing activity. However, the effect of chronic infusion of ghrelin on food consumption and body weight and their possible mechanisms have not been elucidated. In this study, we determined the effects of chronic intracerebroventricular treatment with ghrelin on metabolic factors and on neuropeptide genes that are expressed in hypothalamic neurons that have been previously shown to express the GHS-R and to regulate food consumption. Chronic central administration of rat ghrelin (1 microg/rat every 12 h for 72 h) significantly increased food intake and body weight. However, it did not affect plasma insulin, glucose, leptin, or GH concentrations. We also found that chronic central administration of ghrelin increased both neuropeptide Y (NPY) mRNA levels (151.0 +/- 10.1% of saline-treated controls; P < 0.05) and AGRP mRNA levels (160.0 +/- 22.5% of saline-treated controls; P < 0.05) in the arcuate nucleus. Thus, the primary hypothalamic targets of ghrelin are NPY/AGRP-containing neurons, and ghrelin is a newly discovered orexigenic peptide in the brain and stomach.

  7. Polymorphisms of Saccharomyces cerevisiae genes involved in wine production.

    PubMed

    Vigentini, Ileana; Fracassetti, Daniela; Picozzi, Claudia; Foschino, Roberto

    2009-03-01

    The setting up of new molecular methods for Saccharomyces cerevisiae typing is valuable in enology. Actually, the ability to discriminate different strains in wine making can have a benefit both for the control of the fermentation process and for the preservation of wine typicity. This study focused on the screening of single-nucleotide polymorphisms in genes involved in wine production that could evolve rapidly considering the selective pressure of the isolation environment. Preliminary screening of 30 genes in silico was performed, followed by the selection of 10 loci belonging to 8 genes. The sequence analysis showed a low polymorphism and a degree of heterozygosity. However, a new potential molecular target was recognized in the TPS1 gene coding for the trehalose-6-phosphate synthase enzyme involved in the ethanol resistance mechanism. This gene showed a 1.42% sequence diversity with seven different nucleotide substitutions. Moreover, classic techniques were applied to a collection of 50 S. cerevisiae isolates, mostly with enologic origin. Our results confirmed that the wine making was not carried out only by the inoculated commercial starter because indigenous strains of S. cerevisiae present during fermentation were detected. In addition, a high genetic relationship among some commercial cultures was found, highlighting imprecision or fraudulent practices by starter manufacturers.

  8. Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes.

    PubMed

    Abdel-Ghany, Salah E; Day, Irene; Heuberger, Adam L; Broeckling, Corey D; Reddy, Anireddy S N

    2013-11-01

    1,2,4-butanetriol (butanetriol) is a useful precursor for the synthesis of the energetic material butanetriol trinitrate and several pharmaceutical compounds. Bacterial synthesis of butanetriol from xylose or arabinose takes place in a pathway that requires four enzymes. To produce butanetriol in plants by expressing bacterial enzymes, we cloned native bacterial or codon optimized synthetic genes under different promoters into a binary vector and stably transformed Arabidopsis plants. Transgenic lines expressing introduced genes were analyzed for the production of butanetriol using gas chromatography coupled to mass spectrometry (GC-MS). Soil-grown transgenic plants expressing these genes produced up to 20 µg/g of butanetriol. To test if an exogenous supply of pentose sugar precursors would enhance the butanetriol level, transgenic plants were grown in a medium supplemented with either xylose or arabinose and the amount of butanetriol was quantified. Plants expressing synthetic genes in the arabinose pathway showed up to a forty-fold increase in butanetriol levels after arabinose was added to the medium. Transgenic plants expressing either bacterial or synthetic xylose pathways, or the arabinose pathway showed toxicity symptoms when xylose or arabinose was added to the medium, suggesting that a by-product in the pathway or butanetriol affected plant growth. Furthermore, the metabolite profile of plants expressing arabinose and xylose pathways was altered. Our results demonstrate that bacterial pathways that produce butanetriol can be engineered into plants to produce this chemical. This proof-of-concept study for phytoproduction of butanetriol paves the way to further manipulate metabolic pathways in plants to enhance the level of butanetriol production.

  9. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  10. Histochemical Characterization, Distribution and Morphometric Analysis of NADPH Diaphorase Neurons in the Spinal Cord of the Agouti

    PubMed Central

    Freire, Marco Aurélio M.; Tourinho, Suzane C.; Guimarães, Joanilson S.; Oliveira, Jorge Luiz F.; Picanço-Diniz, Cristovam W.; Gomes-Leal, Walace; Pereira, Antonio

    2008-01-01

    We evaluated the neuropil distribution of the enzymes NADPH diaphorase (NADPH-d) and cytochrome oxidase (CO) in the spinal cord of the agouti, a medium-sized diurnal rodent, together with the distribution pattern and morphometrical characteristics of NADPH-d reactive neurons across different spinal segments. Neuropil labeling pattern was remarkably similar for both enzymes in coronal sections: reactivity was higher in regions involved with pain processing. We found two distinct types of NADPH-d reactive neurons in the agouti's spinal cord: type I neurons had large, heavily stained cell bodies while type II neurons displayed relatively small and poorly stained somata. We concentrated our analysis on type I neurons. These were found mainly in the dorsal horn and around the central canal of every spinal segment, with a few scattered neurons located in the ventral horn of both cervical and lumbar regions. Overall, type I neurons were more numerous in the cervical region. Type I neurons were also found in the white matter, particularly in the ventral funiculum. Morphometrical analysis revealed that type I neurons located in the cervical region have dendritic trees that are more complex than those located in both lumbar and thoracic regions. In addition, NADPH-d cells located in the ventral horn had a larger cell body, especially in lumbar segments. The resulting pattern of cell body and neuropil distribution is in accordance with proposed schemes of segregation of function in the mammalian spinal cord. PMID:18958200

  11. Identification of the neurofibromatosis type 1 gene product

    SciTech Connect

    Gutmann, D.H.; Wood, D.L.; Collins, F.S. )

    1991-11-01

    The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of {approx}250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to the synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested.

  12. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    PubMed Central

    Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M.; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways. PMID:25368184

  13. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    SciTech Connect

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  14. Alphavirus vectors: applications for DNA vaccine production and gene expression.

    PubMed

    Lundstrom, K

    2000-01-01

    Replication-deficient alphavirus vectors have been developed for efficient high-level transgene expression. The broad host range of alphaviruses has allowed infection of a wide variety of mammalian cell lines and primary cultures. Particularly, G protein-coupled receptors have been expressed at high levels and subjected to binding and functional studies. Expression in suspension cultures has greatly facilitated production of large quantities of recombinant proteins for structural studies. Injection of recombinant alphavirus vectors into rodent brain resulted in local reporter gene expression. Highly neuron-specific expression was obtained in hippocampal slice cultures in vivo. Additionally, preliminary studies in animal models suggest that alphavirus vectors can be attractive candidates for gene therapy applications. Traditionally alphavirus vectors, either attenuated strains or replication-deficient particles, have been used to elicit efficient immune responses in animals. Recently, the application of alphaviruses has been extended to naked nucleic acids. Injection of DNA as well as RNA vectors has demonstrated efficient antigen production. In many cases, protection against lethal challenges has been obtained after immunization with alphavirus particles or nucleic acid vectors. Alphavirus vectors can therefore be considered as potentially promising vectors for vaccine production.

  15. Single gene insertion drives bioalcohol production by a thermophilic archaeon.

    PubMed

    Basen, Mirko; Schut, Gerrit J; Nguyen, Diep M; Lipscomb, Gina L; Benn, Robert A; Prybol, Cameron J; Vaccaro, Brian J; Poole, Farris L; Kelly, Robert M; Adams, Michael W W

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  16. 76 FR 9028 - Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-16

    ... Industry: Potency Tests for Cellular and Gene Therapy Products'' dated January 2011. The guidance document provides manufacturers of cellular and gene therapy (CGT) products with recommendations for developing... document entitled ``Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products''...

  17. Regulation of photoreceptor gene expression by the retinal homeobox (Rx) gene product

    PubMed Central

    Pan, Yi; Martinez-De Luna, Reyna I.; Lou, Chih-Hong; Nekkalapudi, Srivamsi; Kelly, Lisa E.; Sater, Amy K.; El-Hodiri, Heithem M.

    2010-01-01

    The retinal homeobox (Rx) gene product is essential for eye development. However little is known about its molecular function. It has been demonstrated that Rx binds to photoreceptor conserved element (PCE-1), a highly conserved element found in the promoter region of photoreceptor-specific genes such as rhodopsin and red cone opsin. We verify that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx binds to the rhodopsin and red cone opsin promoters in vivo. We also find that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Finally, we demonstrate that reduction of Rx expression in tadpoles results in decreases in expression of several PCE-1 containing photoreceptor genes, abnormal photoreceptor morphology, and impaired vision. Our data suggests that Rx, in combination with other transcription factors, is necessary for normal photoreceptor gene expression, maintenance, and function. This establishes a direct role for Rx in regulation of genes expressed in a differentiated cell type. PMID:20060393

  18. Vitreoscilla hemoglobin gene ( vgb) improves lutein production in Chlorella vulgaris

    NASA Astrophysics Data System (ADS)

    Ma, Ruijuan; Lin, Xiangzhi

    2014-03-01

    Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the efficiency of cell respiration and metabolism. In this study, we introduced a Vitreoscilla hemoglobin gene ( vgb) into Chlorella vulgaris by Agrobacterium tumefaciens -mediated transformation (ATMT). PCR analysis confirmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome. Analysis of biomass obtained in shake flasks revealed transformant biomass concentrations as high as 3.28 g/L, which was 38.81% higher than that of the wild-type strain. Lutein content of transformants also increased slightly. Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants, which was 36.77% higher than that of the wild-type strain. The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris, with applications to lutein production from Chlorella during fermentation.

  19. Polyhydroxyalkanoate production in Rhodobacter capsulatus: genes, mutants, expression, and physiology.

    PubMed Central

    Kranz, R G; Gabbert, K K; Locke, T A; Madigan, M T

    1997-01-01

    Like many other prokaryotes, the photosynthetic bacterium Rhodobacter capsulatus produces high levels of polyhydroxyalkanoates (PHAs) when a suitable carbon source is available. The three genes that are traditionally considered to be necessary in the PHA biosynthetic pathway, phaA (beta-ketothiolase), phaB (acetoacetylcoenzyme A reductase), and phaC (PHA synthase), were cloned from Rhodobacter capsulatus. In R. capsulatus, the phaAB genes are not linked to the phaC gene. Translational beta-galactosidase fusions to phaA and phaC were constructed and recombined into the chromosome. Both phaC and phaA were constitutively expressed regardless of whether PHA production was induced, suggesting that control is posttranslational at the enzymatic level. Consistent with this conclusion, it was shown that the R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus grown on numerous carbon sources were determined, indicating that this bacterium grows on C2 to C12 fatty acids. Grown on acetone, caproate, or heptanoate, wild-type R. capsulatus produced high levels of PHAs. Although a phaC deletion strain was unable to synthesize PHAs on any carbon source, phaA and phaAB deletion strains were able to produce PHAs, indicating that alternative routes for the synthesis of substrates for the synthase are present. The nutritional versatility and bioenergetic versatility of R. capsulatus, coupled with its ability to produce large amounts of PHAs and its genetic tractability, make it an attractive model for the study of PHA production. PMID:9251189

  20. Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine.

    PubMed Central

    Plamann, M D; Stauffer, G V

    1989-01-01

    The methionine component of glyA gene regulation in Escherichia coli K-12 was investigated. The results indicate that the glyA gene is positively controlled by the metR gene product. Activation of glyA by the MetR protein requires homocysteine, an intermediate in methionine biosynthesis. The positive-acting metR regulatory system functions independently of a regulatory system shown previously to control glyA gene expression. PMID:2670901

  1. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    DOEpatents

    Wohlbach, Dana J.; Gasch, Audrey P.

    2014-08-05

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  2. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    SciTech Connect

    Wohlbach, Dana J.; Gasch, Audrey P.

    2015-09-29

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  3. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    DOEpatents

    Wohlbach, Dana J.; Gasch, Audrey P.

    2016-11-29

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  4. Identification of the dnaA and dnaN gene products of Escherichia coli.

    PubMed

    Yuasa, S; Sakakibara, Y

    1980-01-01

    A specialized transducing lambda phage carrying the dnaN genes of Escherichia coli specifies two proteins of about 41 and 48 kilodaltons (kd). The temperature-sensitive mutations, dnaN59 and dnaA167, were found to result in altered isoelectric points of the 41 and 48 kd proteins, respectively. Thus the dnaN gene product was identified as a weakly acidic 41 and 48 kd protein. The synthesis of the dnaN gene product is greatly reduced by insertion of a transposon Tn3 in the dnaA gene and by deletion in the gene at the distal end to the dnaN gene. Temperature-sensitive dnaA mutations, on the dnaN gene product. These results indicate that the synthesis of the dnaN gene product is dependent on the structural integrity of the dnaA gene.

  5. Callosal axon arbors in the limb representations of the somatosensory cortex (SI) in the agouti (Dasyprocta primnolopha).

    PubMed

    Rocha, E G; Santiago, L F; Freire, M A M; Gomes-Leal, W; Dias, I A; Lent, R; Houzel, J C; Franca, J G; Pereira, A; Picanço-Diniz, C W

    2007-01-10

    The present report compares the morphology of callosal axon arbors projecting from and to the hind- or forelimb representations in the primary somatosensory cortex (SI) of the agouti (Dasyprocta primnolopha), a large, lisencephlic Brazilian rodent that uses forelimb coordination for feeding. Callosal axons were labeled after single pressure (n = 6) or iontophoretic injections (n = 2) of the neuronal tracer biotinylated dextran amine (BDA, 10 kD), either into the hind- (n = 4) or forelimb (n = 4) representations of SI, as identified by electrophysiological recording. Sixty-nine labeled axon fragments located across all layers of contralateral SI representations of the hindlimb (n = 35) and forelimb (n = 34) were analyzed. Quantitative morphometric features such as densities of branching points and boutons, segments length, branching angles, and terminal field areas were measured. Cluster analysis of these values revealed the existence of two types of axon terminals: Type I (46.4%), less branched and more widespread, and Type II (53.6%), more branched and compact. Both axon types were asymmetrically distributed; Type I axonal fragments being more frequent in hindlimb (71.9%) vs. forelimb (28.13%) representation, while most of Type II axonal arbors were found in the forelimb representation (67.56%). We concluded that the sets of callosal axon connecting fore- and hindlimb regions in SI are morphometrically distinct from each other. As callosal projections in somatosensory and motor cortices seem to be essential for bimanual interaction, we suggest that the morphological specialization of callosal axons in SI of the agouti may be correlated with this particular function.

  6. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  7. Hyperexpression of two Aspergillus Niger Xylanase Genes in Escherichia Coli and Characterization of the Gene Products

    PubMed Central

    Yi, Xiuli; Shi, Yan; Xu, Hui; Li, Wei; Xie, Jie; Yu, Rongqing; Zhu, Jun; Cao, Yi; Qiao, Dairong

    2010-01-01

    The analysis of individual gene product should enable to clarify the role of a particular enzyme in a complex xylanase system of A. niger. The two genes encoding precursors of co-produced endo-1,4-β-D-xylanases, xynA1 and xynB, were isolated from Aspergillus niger SCTCC 400264 (SCTCC, China) by using RT-PCR technique and then successfully expressed in Escherichia coli BL21. The nucleotide sequences of the xynA1 and xynB genes revealed that they were only 52.5% homology to each other. Characterization of the recombinant enzymes revealed the different properties: the specific activity of recombinant XYNA1 was 16.58 U/mg compared to 1201.7 U/mg for recombinant XYNB; The optimum temperature and pH of the recombinant XYNA1 were 35 °C and 3.0, respectively, whereas the corresponding values for the recombinant XYNB were 55 °C and 5.0, respectively; The recombinant XYNB showed much more thermostability than recombinant XYNA1; The recombinant XYNB showed 94% of maximal activity after incubating in water for 60 min at 60 °C compared to no activity for recombinant XYNA1. Various metal ions had different effects on activity between the two recombinant xylanases. PMID:24031555

  8. The Wilms’ Tumor Suppressor Gene (wt1) Product Regulates Dax-1 Gene Expression during Gonadal Differentiation

    PubMed Central

    Kim, Jungho; Prawitt, Dirk; Bardeesy, Nabeel; Torban, Elena; Vicaner, Caroline; Goodyer, Paul; Zabel, Bernard; Pelletier, Jerry

    1999-01-01

    Gonadal differentiation is dependent upon a molecular cascade responsible for ovarian or testicular development from the bipotential gonadal ridge. Genetic analysis has implicated a number of gene products essential for this process, which include Sry, WT1, SF-1, and DAX-1. We have sought to better define the role of WT1 in this process by identifying downstream targets of WT1 during normal gonadal development. We have noticed that in the developing murine gonadal ridge, wt1 expression precedes expression of Dax-1, a nuclear receptor gene. We document here that the spatial distribution profiles of both proteins in the developing gonad overlap. We also demonstrate that WT1 can activate the Dax-1 promoter. Footprinting analysis, transient transfections, promoter mutagenesis, and mobility shift assays suggest that WT1 regulates Dax-1 via GC-rich binding sites found upstream of the Dax-1 TATA box. We show that two WT1-interacting proteins, the product of a Denys-Drash syndrome allele of wt1 and prostate apoptosis response-4 protein, inhibit WT1-mediated transactivation of Dax-1. In addition, we demonstrate that WT1 can activate the endogenous Dax-1 promoter. Our results indicate that the WT1–DAX-1 pathway is an early event in the process of mammalian sex determination. PMID:10022915

  9. Production and clinical development of nanoparticles for gene delivery

    PubMed Central

    Chen, Jie; Guo, Zhaopei; Tian, Huayu; Chen, Xuesi

    2016-01-01

    Gene therapy is a promising strategy for specific treatment of numerous gene-associated human diseases by intentionally altering the gene expression in pathological cells. A successful clinical application of gene-based therapy depends on an efficient gene delivery system. Many efforts have been attempted to improve the safety and efficiency of gene-based therapies. Nanoparticles have been proved to be the most promising vehicles for clinical gene therapy due to their tunable size, shape, surface, and biological behaviors. In this review, the clinical development of nanoparticles for gene delivery will be particularly highlighted. Several promising candidates, which are closest to clinical applications, will be briefly reviewed. Then, the recent developments of nanoparticles for clinical gene therapy will be identified and summarized. Finally, the development of nanoparticles for clinical gene delivery in future will be prospected. PMID:27088105

  10. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    PubMed

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered.

  11. [Collaborative study on regulatory science for facilitating clinical development of gene therapy products for genetic diseases].

    PubMed

    Uchida, Eriko; Igarashi, Yuka; Sato, Yoji

    2014-01-01

    Gene therapy products are expected as innovative medicinal products for intractable diseases such as life-threatening genetic diseases and cancer. Recently, clinical developments by pharmaceutical companies are accelerated in Europe and the United States, and the first gene therapy product in advanced countries was approved for marketing authorization by the European Commission in 2012. On the other hand, more than 40 clinical studies for gene therapy have been completed or ongoing in Japan, most of them are conducted as clinical researches by academic institutes, and few clinical trials have been conducted for approval of gene therapy products. In order to promote the development of gene therapy products, revision of the current guideline and/or preparation of concept paper to address the evaluation of the quality and safety of gene therapy products are necessary and desired to clearly show what data should be submitted before First-in-Human clinical trials of novel gene therapy products. We started collaborative study with academia and regulatory agency to promote regulatory science toward clinical development of gene therapy products for genetic diseases based on lentivirus and adeno-associated virus vectors; National Center for Child Health and Development (NCCHD), Nippon Medical School and PMDA have been joined in the task force. At first, we are preparing pre-draft of the revision of the current gene therapy guidelines in this project.

  12. The morphology of the pineal gland of the yellow-toothed cavy (Galea Spixii Wagler, 1831) and red-rumped agouti (Dasyprocta leporina linnaeus, 1758).

    PubMed

    Câmara, Felipe Venceslau; Lopes, Igor Renno Guimarães; de Oliveira, Gleidson Benevides; Bezerra, Ferdinando Vinicius Fernandes; de Oliveira, Radan Elvis Matias; Oliveira Júnior, Carlos Magno; Silva, Alexandre Rodrigues; de Oliveira, Moacir Franco

    2015-08-01

    The pineal gland is an endocrine gland found in all mammals. This article describes the morphology of this important gland in two species of Caviideae, namely the yellow-toothed cavy and the red-rumped agouti. Ten adult animals of the two species used in current analysis were retrieved from the Center for the Multiplication of Wild Animals (CEMAS/UFERSA) and euthanized. The glands were removed and photographed in situ and ex situ. They were fixed in a paraformaldehyde solution 4% or glutaraldehyde 2.5% solution and submitted to routine histological techniques respectively for light and scanning electron microscopy. Macroscopically, the pineal gland with its elongated structure may be found between the cerebral hemispheres facing the rostral colliculi. Microscopically, pinealocytes and some glia cells were predominant. Contrastingly, to the cavy's pineal gland, a capsule covered the organ in the agouti, with the emission of incomplete septa to the interior, which divided it into two lobules. Light and scanning electron microscopes failed to show calcareous concretions in the pineal gland. Based on the topography of the cavy's and agouti's pineal gland, it may be classified as supra-callosum and ABC type.

  13. Form gene clustering method about pan-ethnic-group products based on emotional semantic

    NASA Astrophysics Data System (ADS)

    Chen, Dengkai; Ding, Jingjing; Gao, Minzhuo; Ma, Danping; Liu, Donghui

    2016-09-01

    The use of pan-ethnic-group products form knowledge primarily depends on a designer's subjective experience without user participation. The majority of studies primarily focus on the detection of the perceptual demands of consumers from the target product category. A pan-ethnic-group products form gene clustering method based on emotional semantic is constructed. Consumers' perceptual images of the pan-ethnic-group products are obtained by means of product form gene extraction and coding and computer aided product form clustering technology. A case of form gene clustering about the typical pan-ethnic-group products is investigated which indicates that the method is feasible. This paper opens up a new direction for the future development of product form design which improves the agility of product design process in the era of Industry 4.0.

  14. Regulatory Oversight of Gene Therapy and Cell Therapy Products in Korea.

    PubMed

    Choi, Minjoung; Han, Euiri; Lee, Sunmi; Kim, Taegyun; Shin, Won

    2015-01-01

    The Ministry of Food and Drug Safety regulates gene therapy and cell therapy products as biological products under the authority of the Pharmaceutical Affairs Act. As with other medicinal products, gene therapy and cell therapy products are subject to approval for use in clinical trials and for a subsequent marketing authorization and to post-market surveillance. Research and development of gene therapy and cell therapy products have been progressing rapidly in Korea with extensive investment, offering great potential for the treatment of various serious diseases. To facilitate development of safe and effective products and provide more opportunities to patients suffering from severe diseases, several regulatory programs, such as the use of investigational products for emergency situations, fast-track approval, prereview of application packages, and intensive regulatory consultation, can be applied to these products. The regulatory approach for these innovative products is case by case and founded on science-based review that is flexible and balances the risks and benefits.

  15. Requirements for Clinical Trials with Gene Therapy and Transplant Products in Switzerland.

    PubMed

    Marti, Andreas

    2015-01-01

    This chapter aims to describe and summarize the regulation of gene and cell therapy products in Switzerland and its legal basis. Product types are briefly described, as are Swiss-specific terminologies such as the term "transplant product," which means products manufactured from cells, tissues, or even whole organs. Although some parts of this chapter may show a guideline character, they are not legally binding, but represent the current thinking of Swissmedic, the Swiss Agency for Therapeutic Products. As so far the experience with marketing approval of gene therapy and cell therapy products in Switzerland is limited, this chapter focuses on the regulation of clinical trials conducted with these products. Quality, nonclinical, and clinical aspects are summarized separately for gene therapy products and transplant products.

  16. Linking gene regulation to mRNA production and export.

    PubMed

    Rodríguez-Navarro, Susana; Hurt, Ed

    2011-06-01

    Regulation of gene expression can occur at many different levels. One important step in the gene expression process is the transport of mRNA from the nucleus to the cytoplasm. In recent years, studies have described how nuclear mRNA export depends on the steps preceding and following transport through nuclear pore complexes. These include gene activation, transcription, mRNA processing and mRNP assembly and disassembly. In this review, we summarise recent insights into the links between these steps in the gene expression cascade.

  17. Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

    PubMed Central

    Heath, D G; Cleary, P P

    1989-01-01

    The partial nucleotide sequence for an Fc-receptor gene from an M-type 76 group A streptococcus was determined. DNA sequence analysis revealed considerable sequence similarity between the Fc-receptor and M-protein genes in their proposed promoter regions, signal sequences, and 3' termini. Additional analysis indicated that the deduced Fc-receptor protein contains a proline-rich region and membrane anchor region highly similar to that of M protein. In view of these results, we postulated that Fc-receptor and M-protein genes of group A streptococci are the products of gene duplication from a common ancestral gene. It is proposed that DNA sequence similarity between these two genes may allow for extragenic homologous recombination as a means of generating antigenic diversity in these two surface proteins. PMID:2660147

  18. The Spinal Muscular Atrophy Disease Gene Product, Smn

    PubMed Central

    Carvalho, Teresa; Almeida, Fátima; Calapez, Alexandre; Lafarga, Miguel; Berciano, Maria T.; Carmo-Fonseca, Maria

    1999-01-01

    The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called “gemini of coiled bodies” or “gems.” An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway. PMID:10562276

  19. PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

    PubMed Central

    Cao, Yongheng; Nakata, Masanori; Okamoto, Shiki; Takano, Eisuke; Yada, Toshihiko; Minokoshi, Yasuhiko; Hirata, Yukio; Nakajima, Kazunori; Iskandar, Kristy; Hayashi, Yoshitake; Ogawa, Wataru; Barsh, Gregory S.; Hosoda, Hiroshi; Kangawa, Kenji; Itoh, Hiroshi; Noda, Tetsuo; Kasuga, Masato; Nakae, Jun

    2011-01-01

    Insulin and leptin intracellular signaling pathways converge and act synergistically on the hypothalamic phosphatidylinositol-3-OH kinase/3-phosphoinositide-dependent protein kinase 1 (PDK1). However, little is known about whether PDK1 in agouti-related peptide (AGRP) neurons contributes to energy homeostasis. We generated AGRP neuron-specific PDK1 knockout (AGRPPdk1−/−) mice and mice with selective expression of transactivation-defective Foxo1 (Δ256Foxo1AGRPPdk1−/−). The AGRPPdk1−/− mice showed reductions in food intake, body length, and body weight. The Δ256Foxo1AGRPPdk1−/− mice showed increased body weight, food intake, and reduced locomotor activity. After four weeks of calorie-restricted feeding, oxygen consumption and locomotor activity were elevated in AGRPPdk1−/− mice and reduced in Δ256Foxo1AGRPPdk1−/− mice. In vitro, ghrelin-induced changes in [Ca2+]i and inhibition of ghrelin by leptin were significantly attenuated in AGRPPdk1−/− neurons compared to control neurons. However, ghrelin-induced [Ca2+]i changes and leptin inhibition were restored in Δ256Foxo1AGRPPdk1−/− mice. These results suggested that PDK1 and Foxo1 signaling pathways play important roles in the control of energy homeostasis through AGRP-independent mechanisms. PMID:21694754

  20. Agouti Related Peptide Secreted Via Human Mesenchymal Stem Cells Upregulates Proteasome Activity in an Alzheimer’s Disease Model

    PubMed Central

    Lee, Na Kyung; Park, Sang Eon; Kwon, Soo Jin; Shim, Sangmi; Byeon, Yeji; Kim, Jong-Hwa; Na, Duk L.; Chang, Jong Wook

    2017-01-01

    The activity of the ubiquitin proteasome system (UPS) is downregulated in aggregation diseases such as Alzheimer’s disease (AD). In this study, we investigated the therapeutic potential of the Agouti-related peptide (AgRP), which is secreted by human mesenchymal stem cells (MSCs), in terms of its effect on the regulation of proteasome activity in AD. When SH-SY5Y human neuroblastoma cells were co-cultured with MSCs isolated from human Wharton’s Jelly (WJ-MSC), their proteasome activity was significantly upregulated. Further analysis of the conditioned media after co-culture allowed us to identify significant concentrations of a neuropeptide, called AgRP. The stereotactic delivery of either WJ-MSCs or AgRP into the hippocampi of C57BL6/J and 5XFAD mice induced a significant increase of proteasome activity and suppressed the accumulation of ubiquitin-conjugated proteins. Collectively, these findings suggest strong therapeutic potential for WJ-MSCs and AgRP to enhance proteasome activity, thereby potentially reducing abnormal protein aggregation and delaying the clinical progression of various neurodegenerative diseases. PMID:28051110

  1. Identification of potentially hazardous human gene products in GMO risk assessment.

    PubMed

    Bergmans, Hans; Logie, Colin; Van Maanen, Kees; Hermsen, Harm; Meredyth, Michelle; Van Der Vlugt, Cécile

    2008-01-01

    Genetically modified organisms (GMOs), e.g. viral vectors, could threaten the environment if by their release they spread hazardous gene products. Even in contained use, to prevent adverse consequences, viral vectors carrying genes from mammals or humans should be especially scrutinized as to whether gene products that they synthesize could be hazardous in their new context. Examples of such potentially hazardous gene products (PHGPs) are: protein toxins, products of dominant alleles that have a role in hereditary diseases, gene products and sequences involved in genome rearrangements, gene products involved in immunomodulation or with an endocrine function, gene products involved in apoptosis, activated proto-oncogenes. For contained use of a GMO that carries a construct encoding a PHGP, the precautionary principle dictates that safety measures should be applied on a "worst case" basis, until the risks of the specific case have been assessed. The potential hazard of cloned genes can be estimated before empirical data on the actual GMO become available. Preliminary data may be used to focus hazard identification and risk assessment. Both predictive and empirical data may also help to identify what further information is needed to assess the risk of the GMO. A two-step approach, whereby a PHGP is evaluated for its conceptual dangers, then checked by data bank searches, is delineated here.

  2. Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes.

    PubMed

    Li, Chunliang; Qi, Rong; Singleterry, Rebecca; Hyle, Judith; Balch, Amanda; Li, Xiuling; Sublett, Jack; Berns, Hartmut; Valentine, Marcus; Valentine, Virginia; Sherr, Charles J

    2014-05-01

    Injection of transcription activator-like effector nucleases (TALEN) mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescence visualization of phosphorylated histone 2A (γH2AX) combined with fluorescence in situ hybridization revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within 6 h of injection, an activity that continued at reduced efficiency in two-cell embryos. TALEN-Agouti mRNAs injected into zygotes of brown FvB × C57BL/6 hybrid mice generated completely black pups, confirming that mutations were induced prior to, and/or early after, cell division. Founder mice, many of which were mosaic, transmitted altered Agouti alleles to F1 pups to yield an allelic series of mutant strains. Although mutations were targeted to "spacer" sequences flanked by TALEN binding sites, larger deletions that extended beyond the TALEN-binding sequences were also detected and were similarly inherited through the germ line. Zygotic coinjection of TALEN mRNAs directed to the Agouti, miR-205, and the Arf tumor suppressor loci yielded pups containing frequent and heritable mutations of two or three genes. Simultaneous gene editing in zygotes affords an efficient approach for producing mice with compound mutant phenotypes, bypassing constraints of conventional mouse knockout technology in embryonic stem cells.

  3. Vector systems for prenatal gene therapy: principles of non-viral vector design and production.

    PubMed

    Wong, Suet Ping; Argyros, Orestis; Harbottle, Richard P

    2012-01-01

    Gene therapy vectors based on viruses are the most effective gene delivery systems in use today and although efficient at gene transfer their potential toxicity (Hacein-Bey-Abina et al., Science 302:415-419, 2003) provides impetus for the development of safer non-viral alternatives. An ideal vector for human gene therapy should deliver sustainable therapeutic levels of gene expression without affecting the viability of the host at either the cellular or somatic level. Vectors, which comprise entirely human elements, may provide the most suitable method of achieving this. Non-viral vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production, and great versatility. The development of more efficient, economically prepared, and safer gene delivery vectors is a crucial prerequisite for their successful clinical application and remains a primary strategic task of gene therapy research.

  4. Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters.

    PubMed

    Du, Deyao; Zhu, Yu; Wei, Junhong; Tian, Yuqing; Niu, Guoqing; Tan, Huarong

    2013-07-01

    Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.

  5. Proopiomelanocortin, agouti-related protein, and leptin in human cerebrospinal fluid: correlations with body weight and adiposity

    PubMed Central

    Page-Wilson, Gabrielle; Meece, Kana; White, Anne; Rosenbaum, Michael; Leibel, Rudolph L.; Smiley, Richard

    2015-01-01

    Leptin and its neuronal targets, which produce proopiomelanocortin (POMC) and agouti-related protein (AgRP), regulate energy balance. This study characterized leptin, POMC, and AgRP in the cerebrospinal fluid (CSF) of 47 healthy human subjects, 23 lean and 24 overweight/obese (OW/OB), as related to BMI, adiposity, plasma leptin, soluble leptin receptor (s-OB-R), and insulin. POMC was measured since the POMC prohormone is the predominant POMC peptide in CSF and correlates with hypothalamic POMC in rodents. Plasma AgRP was similarly characterized. CSF leptin was 83-fold lower than in plasma and correlated strongly with BMI, body fat, and insulin. The relative amount of leptin transported into CSF declined with increasing BMI, ranging from 4.5 to 0.52%, consistent with a saturable transport mechanism. CSF sOB-R was 78-fold lower than in plasma and correlated negatively with plasma and CSF leptin. CSF POMC was higher in lean vs. OW/OB subjects (P < 0.001) and correlated negatively with CSF leptin (r = −0.60, P < 0.001) and with plasma leptin, insulin, BMI, and adiposity. CSF AgRP was not different in lean vs. OW/OB; however, plasma AgRP was higher in lean subjects (P = 0.001) and correlated negatively with BMI, adiposity, leptin, insulin, and HOMA (P < 0.005). Thus, CSF measurements may provide useful biomarkers for brain leptin and POMC activity. The striking negative correlation between CSF leptin and POMC could be secondary to leptin resistance and/or neuronal changes associated with obesity but may also indicate that POMC plays a primary role in regulating body weight and adiposity. The role of plasma AgRP as a neuroendocrine biomarker deserves further study. PMID:26152765

  6. Acute and long-term effects of a single dose of MDMA on aggression in Dark Agouti rats.

    PubMed

    Kirilly, Eszter; Benko, Anita; Ferrington, Linda; Ando, Romeo D; Kelly, Paul A T; Bagdy, Gyorgy

    2006-02-01

    MDMA causes selective depletion of serotonergic terminals in experimental animals and the consequent decrease in synaptic 5-HT may, inter alia, increase impulsivity. To study the effects of MDMA upon brain function, the behaviour of male Dark Agouti rats exposed to MDMA (15 mg/kg i.p.), two 5-HT1B agonists (CGS-12066A and CP-94,253, both 5 mg/kg i.p.) or saline were investigated in the resident-intruder test. Studies were performed in drug-naive rats and also in rats exposed to MDMA (15 mg/kg i.p.) 21 d earlier. In parallel experiments the functional neuroanatomy of MDMA effects were assessed using 2-deoxyglucose imaging of local cerebral metabolic rate of glucose utilization (LCMRGlu) and neurotoxicity was assessed by measuring [3H]paroxetine binding. There was no significant difference in aggressive behaviour (biting, boxing, wrestling and their latencies) between drug-naive rats and rats previously exposed to MDMA 21 d earlier, despite reduced social behaviour, decreased LCMRGlu in several brain areas involved in aggression, and reductions in paroxetine binding by 30-60% in the forebrain. CGS-12066A, CP-94,253 and acute MDMA produced marked decreases in aggressive behaviours, especially in biting, boxing and kicking found in drug-naive rats. In animals previously exposed to the drug, acute anti-aggressive effects of MDMA were, in general, preserved as were MDMA-induced increases in LCMRGlu. Our studies provide evidence that in the resident-intruder test, where social isolation is a requirement, aggressive behaviour and acute anti-aggressive effects of MDMA and 5-HT1B receptor agonists remain intact 3 wk after a single dose of the drug despite significant damage to the serotonergic system.

  7. Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis

    PubMed Central

    Morlino, Giovanni B.; Tizzani, Lorenza; Fleer, Reinhard; Frontali, Laura; Bianchi, Michele M.

    1999-01-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system. PMID:10543790

  8. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

    PubMed

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M

    1999-11-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  9. DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17.

    PubMed Central

    Genilloud, O; Moreno, F; Kolter, R

    1989-01-01

    The 3.8-kilobase segment of plasmid DNA that contains the genes required for production of the DNA replication inhibitor microcin B17 was sequenced. The sequence contains four open reading frames which were shown to be translated in vivo by the construction of fusions to lacZ. The location of these open reading frames fits well with the location of the four microcin B17 production genes, mcbABCD, identified previously through genetic complementation. The products of the four genes have been identified, and the observed molecular weights of the proteins agree with those predicted from the nucleotide sequence. The transcription of these genes was studied by using fusions to lacZ and physical mapping of mRNA start sites. Three promoters were identified in this region. The major promoter for all the genes is a growth phase-regulated OmpR-dependent promoter located upstream of mcbA. A second promoter is located within mcbC and is responsible for a low-level basal expression of mcbD. A third promoter, located within mcbD, promotes transcription in the reverse direction starting within mcbD and extending through mcbC. The resulting mRNA appears to be an untranslated antisense transcript that could play a regulatory role in the expression of these genes. Images PMID:2644225

  10. Organogenesis and foetal haemodynamics during the normal gestation of healthy black-rumped agoutis (Dasyprocta prymnolopha, Wagler, 1831) bred in captivity.

    PubMed

    Sousa, Fca; Pessoa, G T; Moura, L S; Araújo, J R; Rodrigues, Rps; Barbosa, Maps; Diniz, A N; Souza, A B; Silva, E G; Lucena, L U; Sanches, M P; Silva-Filho, O F; Guerra, P C; Sousa, J M; Neves, W C; Alves, F R

    2017-02-01

    The objective of this study was to define the patterns of organogenesis and foetal haemodynamics during the normal gestation of healthy agoutis (Dasyprocta prymnolopha) kept in captivity. Thirty pregnant agoutis that ranged in size from small to medium and weighed between 2.5 and 3 kg underwent B-mode and Doppler ultrasonography for the biometric evaluation of the foetal organs. The foetal aortic blood flow proved to be predominantly systolic, and the measured flow velocity was 78.89 ± 2.95 cm/s, with a maximum pressure gradient of 2.12 ± 0.27 mmHg. The liver was characterized by its large volume, occupying the entire cranial aspect of the abdominal cavity, and it was associated cranially with the diaphragm and caudally with the stomach. The flow velocity in the portal vein was estimated to equal 12.17 ± 2.37 cm/s, with a resistivity index of 0.82 ± 0.05. The gallbladder was centrally located and protruded cranially towards the diaphragm. The spleen was visualized as an elongated structure with tapered cranial and caudal extremities, and the foetal kidneys were visualized bilaterally in the retroperitoneal region, with the right kidney positioned slightly more cranially than the left. The morphological characterization and hemodynamic analysis of the foetal organs of black-rumped agoutis via B-mode and Doppler ultrasonography allow determination of the vascular network and of reference values for the blood flow required for perfusing the anatomical elements essential for maintaining the viability of foetuses at different gestational ages.

  11. Rapid development of semistarvation-induced hyperactivity in Dark Agouti rats. Excessive wheel running and effect of 3,4-methylenedioxymethamphetamine (MDMA).

    PubMed

    Vidal, Pedro; Pérez-Padilla, Ángeles; Pellón, Ricardo

    2013-02-01

    Clinical studies have found that patients with anorexia develop high activity levels. These data suggest a possible implication of activity in the aetiology of anorexia and are in line with findings obtained in animals during experimental procedures to model interactions between activity and weight loss. Activity-based anorexia (ABA) and semistarvation-induced hyperactivity (SIH) develop when laboratory rats have food access restricted to a single period in the day and are given free access to an activity wheel. This experiment sought to show the effect on weight loss of the excessive activity normally seen in Dark Agouti rats and of hyperactivity induced by 3,4-methylenedioxymethamphetamine (MDMA). To this end, 32 female rats of the Dark Agouti strain were selected and divided into four groups in accordance with a 2 × 2 factorial design, in which one factor was treatment (saline or MDMA) and the other was access or lack of access to an activity wheel. Animals with wheel running access displayed a marked increase in running combined with accelerated weight loss. Although pharmacological treatment resulted in no observable effect on weight loss, rats treated with 12.5mg/kg MDMA generally registered more wheel running than did those treated with saline. Analysis of data on the temporal distribution of wheel running revealed an alteration in circadian activity patterns as a consequence of MDMA. These results, by showing a general high level of wheel running in Dark Agouti rats, once again emphasise the close relationship between activity and weight loss in the development of SIH and related phenomena such as ABA.

  12. WWOX gene and gene product: tumor suppression through specific protein interactions.

    PubMed

    Salah, Zaidoun; Aqeilan, Rami; Huebner, Kay

    2010-02-01

    The WWOX gene, an archetypal fragile gene, encompasses a chromosomal fragile site at 16q23.2, and encodes the approximately 46-kDa Wwox protein, with WW domains that interact with a growing list of interesting proteins. If the function of a protein is defined by the company it keeps, then Wwox is involved in numerous important signal pathways for bone and germ-cell development, cellular and animal growth and death, transcriptional control and suppression of cancer development. Because alterations to genes at fragile sites are exquisitely sensitive to replication stress-induced DNA damage, there has been an ongoing scientific discussion questioning whether such gene expression alterations provide a selective advantage for clonal expansion of neoplastic cells, and a parallel discussion on why important genes would be present at sites that are susceptible to inactivation. We offer some answers through a description of known WWOX functions.

  13. Database of cattle candidate genes and genetic markers for milk production and mastitis

    PubMed Central

    Ogorevc, J; Kunej, T; Razpet, A; Dovc, P

    2009-01-01

    A cattle database of candidate genes and genetic markers for milk production and mastitis has been developed to provide an integrated research tool incorporating different types of information supporting a genomic approach to study lactation, udder development and health. The database contains 943 genes and genetic markers involved in mammary gland development and function, representing candidates for further functional studies. The candidate loci were drawn on a genetic map to reveal positional overlaps. For identification of candidate loci, data from seven different research approaches were exploited: (i) gene knockouts or transgenes in mice that result in specific phenotypes associated with mammary gland (143 loci); (ii) cattle QTL for milk production (344) and mastitis related traits (71); (iii) loci with sequence variations that show specific allele-phenotype interactions associated with milk production (24) or mastitis (10) in cattle; (iv) genes with expression profiles associated with milk production (207) or mastitis (107) in cattle or mouse; (v) cattle milk protein genes that exist in different genetic variants (9); (vi) miRNAs expressed in bovine mammary gland (32) and (vii) epigenetically regulated cattle genes associated with mammary gland function (1). Fourty-four genes found by multiple independent analyses were suggested as the most promising candidates and were further in silico analysed for expression levels in lactating mammary gland, genetic variability and top biological functions in functional networks. A miRNA target search for mammary gland expressed miRNAs identified 359 putative binding sites in 3′UTRs of candidate genes. PMID:19508288

  14. Comparative genomics of actinomycetes with a focus on natural product biosynthetic genes

    PubMed Central

    2013-01-01

    Background Actinomycetes are a diverse group of medically, industrially and ecologically important bacteria, studied as much for the diseases they cause as for the cures they hold. The genomes of actinomycetes revealed that these bacteria have a large number of natural product gene clusters, although many of these are difficult to tie to products in the laboratory. Large scale comparisons of these clusters are difficult to perform due to the presence of highly similar repeated domains in the most common biosynthetic machinery: polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). Results We have used comparative genomics to provide an overview of the genomic features of a set of 102 closed genomes from this important group of bacteria with a focus on natural product biosynthetic genes. We have focused on well-represented genera and determine the occurrence of gene cluster families therein. Conservation of natural product gene clusters within Mycobacterium, Streptomyces and Frankia suggest crucial roles for natural products in the biology of each genus. The abundance of natural product classes is also found to vary greatly between genera, revealing underlying patterns that are not yet understood. Conclusions A large-scale analysis of natural product gene clusters presents a useful foundation for hypothesis formulation that is currently underutilized in the field. Such studies will be increasingly necessary to study the diversity and ecology of natural products as the number of genome sequences available continues to grow. PMID:24020438

  15. Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations

    SciTech Connect

    Moore, R.C.; Redhead, N.J.; Selfridge, J.

    1995-09-01

    We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies. 43 refs., 6 figs., 1 tab.

  16. Mobile antibiotic resistance - the spread of genes determining the resistance of bacteria through food products.

    PubMed

    Godziszewska, Jolanta; Guzek, Dominika; Głąbski, Krzysztof; Wierzbicka, Agnieszka

    2016-07-07

    In recent years, more and more antibiotics have become ineffective in the treatment of bacterial nfections. The acquisition of antibiotic resistance by bacteria is associated with circulation of genes in the environment. Determinants of antibiotic resistance may be transferred to pathogenic bacteria. It has been shown that conjugation is one of the key mechanisms responsible for spread of antibiotic resistance genes, which is highly efficient and allows the barrier to restrictions and modifications to be avoided. Some conjugative modules enable the transfer of plasmids even between phylogenetically distant bacterial species. Many scientific reports indicate that food is one of the main reservoirs of these genes. Antibiotic resistance genes have been identified in meat products, milk, fruits and vegetables. The reason for such a wide spread of antibiotic resistance genes is the overuse of antibiotics by breeders of plants and animals, as well as by horizontal gene transfer. It was shown, that resistance determinants located on mobile genetic elements, which are isolated from food products, can easily be transferred to another niche. The antibiotic resistance genes have been in the environment for 30 000 years. Their removal from food products is not possible, but the risks associated with the emergence of multiresistant pathogenic strains are very large. The only option is to control the emergence, selection and spread of these genes. Therefore measures are sought to prevent horizontal transfer of genes. Promising concepts involve the combination of developmental biology, evolution and ecology in the fight against the spread of antibiotic resistance.

  17. Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster.

    PubMed

    Seip, Britta; Galinski, Erwin A; Kurz, Matthias

    2011-02-01

    We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190(T) and evaluate the suitability of P. stutzeri DSM5190(T) for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in E. coli under the conditions employed exceeded that of the natural producer Pseudomonas stutzeri and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.

  18. Polymorphism of the ovocalyxin-32 gene and its association with egg production traits in the chicken.

    PubMed

    Uemoto, Y; Suzuki, C; Sato, S; Sato, S; Ohtake, T; Sasaki, O; Takahashi, H; Kobayashi, E

    2009-12-01

    We performed candidate gene analysis to identify SNP in the chicken ovocalyxin-32 (OCX-32) gene in the F(2) resource population, to develop a PCR-RFLP method for genotyping and to evaluate the associations of the gene polymorphism with egg production traits. The F(2) resource population-comprising 272 chickens-was obtained by crossing White Leghorn (WL) males and Rhode Island Red (RIR) females. They were measured for egg production traits and used for candidate gene analysis. Among parental individuals of the F(2) population, 2 novel nonsynonymous polymorphisms (c.267T>G and c.494A>C) and 1 known nonsynonymous polymorphism (c.381G>C) in the coding sequences of the chicken OCX-32 gene were detected. The PCR-RFLP method was used for screening the chickens of the F(2) population. In parental populations, genotype c.267T>G and c.494A>C were segregated within WL and RIR breeds, respectively, but genotype c.381G>C was breed-specific SNP between WL and RIR breeds. A total of 4 haplotypes were constructed based on the 3 SNP in parental populations, and there was no recombination between c.267T>G and c.494A>C. There was a significant association (P < 0.05) between the OCX-32 gene SNP and egg production traits, but there was no significant association between the haplotypes of the OCX-32 gene and egg production traits in the F(2) population. In the present study, there was the most significant association between c.381G>C of the OCX-32 gene and rate of egg production. The current study is the first step to confirm the relationship between OCX-32 gene polymorphisms and egg production traits.

  19. Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics

    SciTech Connect

    Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. S.; Ahring, Birgitte K.; Linggi, Bryan E.

    2013-06-19

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding the system level regulation and control of the pathway. To address this limitation, we examined Bacillus subtilis grown under multiple conditions and then determined the relationship between altered isoprene production and the pattern of gene expression. We found that terpenoid genes appeared to fall into two distinct subsets with opposing correlations with respect to the amount of isoprene produced. The group whose expression levels positively correlated with isoprene production included dxs, the gene responsible for the commitment step in the pathway, as well as ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. This analysis showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model which accurately predicts production of this secondary metabolite across many simulated environmental conditions.

  20. Antibacterial Discovery and Development: From Gene to Product and Back

    PubMed Central

    Fedorenko, Victor; Genilloud, Olga; Horbal, Liliya; Marcone, Giorgia Letizia; Marinelli, Flavia; Paitan, Yossi; Ron, Eliora Z.

    2015-01-01

    Concern over the reports of antibiotic-resistant bacterial infections in hospitals and in the community has been publicized in the media, accompanied by comments on the risk that we may soon run out of antibiotics as a way to control infectious disease. Infections caused by Enterococcus faecium, Staphylococcus aureus, Klebsiella species, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and other Enterobacteriaceae species represent a major public health burden. Despite the pharmaceutical sector's lack of interest in the topic in the last decade, microbial natural products continue to represent one of the most interesting sources for discovering and developing novel antibacterials. Research in microbial natural product screening and development is currently benefiting from progress that has been made in other related fields (microbial ecology, analytical chemistry, genomics, molecular biology, and synthetic biology). In this paper, we review how novel and classical approaches can be integrated in the current processes for microbial product screening, fermentation, and strain improvement. PMID:26339625

  1. Nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum.

    PubMed

    Nijland, Jeroen G; Ebbendorf, Bjorg; Woszczynska, Marta; Boer, Rémon; Bovenberg, Roel A L; Driessen, Arnold J M

    2010-11-01

    Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the β-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.

  2. Id-1 and Id-2 genes and products as markers of epithelial cancer

    DOEpatents

    Desprez, Pierre-Yves; Campisi, Judith

    2008-09-30

    A method for detection and prognosis of breast cancer and other types of cancer. The method comprises detecting expression, if any, for both an Id-1 and an Id-2 genes, or the ratio thereof, of gene products in samples of breast tissue obtained from a patient. When expressed, Id-1 gene is a prognostic indicator that breast cancer cells are invasive and metastatic, whereas Id-2 gene is a prognostic indicator that breast cancer cells are localized and noninvasive in the breast tissue.

  3. Id-1 and Id-2 genes and products as markers of epithelial cancer

    DOEpatents

    Desprez, Pierre-Yves [El Cerrito, CA; Campisi, Judith [Berkeley, CA

    2011-10-04

    A method for detection and prognosis of breast cancer and other types of cancer. The method comprises detecting expression, if any, for both an Id-1 and an Id-2 genes, or the ratio thereof, of gene products in samples of breast tissue obtained from a patient. When expressed, Id-1 gene is a prognostic indicator that breast cancer cells are invasive and metastatic, whereas Id-2 gene is a prognostic indicator that breast cancer cells are localized and noninvasive in the breast tissue.

  4. B-cell translocation gene 2 promotes hepatic hepcidin production via induction of Yin Yang 1.

    PubMed

    Lee, Sung-Eun; Hwang, Seung-Lark; Jang, Won-Gu; Chang, Hyeun Wook; Kim, Yong Deuk

    2015-05-15

    Hepcidin is a peptide hormone secreted in the liver and plays a key role in maintaining iron homeostasis. Here, we demonstrate that B-cell translocation gene 2 (BTG2) is a key player in hepatic hepcidin regulation via induction of Yin Yang 1 (YY1). Hepatic hepcidin gene expression significantly enhanced by fasting states and glucagon exposure led to induction of gluconeogenic gene expression, and elevated serum hepcidin production in mice. Notably, overexpression of BTG2 using adenoviral system (Ad-BTG2) significantly elevated serum hepcidin levels via a significant induction of YY1 gene transcription. Immunoprecipitation studies demonstrated that BTG2 physically interacted with YY1 and recruited on the hepcidin gene promoter. Finally, ablation of hepatic BTG2 gene by gene silencing markedly attenuated the elevation of serum hepcidin production along with YY1 and hepcidin mRNA expression in fasting state. Likewise, forskolin (FSK)-stimulated hepcidin promoter activity was dramatically disrupted by endogenous BTG2 knockdown. Overall, our current study provides a novel molecular mechanism of BTG2-mediated induction of hepcidin gene expression, thereby contributing to a better understanding of the hepatic hepcidin production involved in iron homeostasis.

  5. Dynamics and modeling of temperature-regulated gene product expression in recombinant yeast fermentation.

    PubMed

    Cheng, C; Yang, S T

    1996-06-20

    The dynamic response of temperature-regulated gene expression in the recombinant yeast Saccharomyces cerevisiae, strain XK1-C2 carrying plasmid pSXR125, to temperature changes during fed-batch and continuous (chemostat) cultures was studied. The production of the gene product, beta-galactosidase, in the yeast cell is sensitive to the growth temperature. Gene expression of this product was fully turned on or off by temperature shifts between 24 and 30 degrees C. However, the response for gene turn-on and turn-off in this recombinant yeast was slow, requiring from several hours to over 10 h to fully appear. The continuous reactor took 30-60 h after the temperature shift to reach a new steady state. A dynamic process model was developed to simulate the reactor and cell responses to temperature shift. A first-order model was used to account for the effect of dilution rate on the change of protein concentration in the chemostat. It was found that cell response in gene expression to temperature shift followed first-order plus dead-time dynamics. Also, the response time for gene expression to temperature shift varied with specific growth rate or dilution rate of the continuous reactor. In general, the response was slower at a higher dilution rate and for gene turn-on than for gene turn-off.

  6. Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production

    NASA Astrophysics Data System (ADS)

    Bailey, Andy M.; Alberti, Fabrizio; Kilaru, Sreedhar; Collins, Catherine M.; de Mattos-Shipley, Kate; Hartley, Amanda J.; Hayes, Patrick; Griffin, Alison; Lazarus, Colin M.; Cox, Russell J.; Willis, Christine L.; O’Dwyer, Karen; Spence, David W.; Foster, Gary D.

    2016-05-01

    Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

  7. Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production

    PubMed Central

    Bailey, Andy M.; Alberti, Fabrizio; Kilaru, Sreedhar; Collins, Catherine M.; de Mattos-Shipley, Kate; Hartley, Amanda J.; Hayes, Patrick; Griffin, Alison; Lazarus, Colin M.; Cox, Russell J.; Willis, Christine L.; O’Dwyer, Karen; Spence, David W.; Foster, Gary D.

    2016-01-01

    Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi. PMID:27143514

  8. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    SciTech Connect

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  9. [Quality control of gene therapy products: approach of the French Agency for the Safety of Health Products].

    PubMed

    Chenivesse, Xavier; Ridoux, Valérie; Tissier, Marie-Hélène

    2003-04-01

    Gene therapy is a new therapeutic strategy which can constitute in some diseases a true alternative or a complement to the "classical treatments". Regarding the innovative features, the complexity and the extreme diversity of the gene therapy products (naked DNA, synthetic vectors, viral vectors, genetically modified cells), these new products presently in clinical trials have to be precisely evaluated and controlled for their medicine quality as well as their biological origin and/or their specific characteristics of genetically modified organisms. The French Agency for the Safety of Health Products engaged an in-depth scientific review concerning the control of this very heterogeneous class of potential therapeutics through the creation of a working group. The objectives of this group were to determine the testings to be performed by a national authority for each type of gene therapy products and to select the appropriate techniques or methods to be developed. Controls considered as essential are listed and include the verification of the identity, the purity, the transfer and expression efficiency as well as the microbiological and viral safety of the products. This implies the development of diverse techniques of molecular biology, cellular biology, physico-chemistry, animal testing, histology and microbiology. Finally, in order to define the basis of testings of these emerging products, the marketing of which should be effective for some of them in the next years, it appears extremely important to harmonize the quality, efficiency and safety criteria, to develop specific references and standards and to create specific guidelines for the control of gene therapy products.

  10. Characterization of a beta-tubulin gene and a beta-tubulin gene products of Brugia pahangi.

    PubMed

    Guénette, S; Prichard, R K; Klein, R D; Matlashewski, G

    1991-02-01

    A genomic clone containing a beta-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the beta-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mNRA of 1.8 kb which codes for a protein of 448 amino acids. The predicted beta-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken beta 2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct beta-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi beta-tubulin was used to identify beta-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilariae and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved beta-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the beta-tubulin isoforms of B. pahangi.

  11. Identification and distribution of products from novel tryptopyrokinin genes in the locust, Locusta migratoria.

    PubMed

    Redeker, Jana; Bläser, Marcel; Neupert, Susanne; Predel, Reinhard

    2017-04-22

    A recent analysis of the genome of Locusta migratoria indicated the presence of four novel insect neuropeptide genes encoding for multiple tryptopyrokinin peptides (tryptoPKs); hitherto only known from pyrokinin or capa genes. In our study, mature products of tryptoPK genes 1 and 2 were identified by mass spectrometry; precursor sequences assigned to the tryptoPK genes 3 and 4 are likely partial sequences of a single precursor. The expression of tryptoPK genes 1 and 2 is restricted to two cells in the subesophageal ganglion, exhibiting not only a unique neuropeptidome but also a very distinctive axonal projection. Comparative neuroendocrinology revealed that homologous cells in other insects also produce tryptoPKs but use other genes to generate this pattern. Since capa and pyrokinin genes are discussed as ancestors of the tryptoPK genes, we completed the hitherto only partially known precursor sequences of these genes by means of transcriptome analyses. The distribution of mature products of CAPA and pyrokinin precursors in the CNS is compared with that of tryptoPKs. In addition, a novel pyrokinin-like precursor is described.

  12. Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics

    PubMed Central

    Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. Steven; Ahring, Birgitte K.; Linggi, Bryan

    2013-01-01

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions. PMID:23840410

  13. Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics.

    PubMed

    Hess, Becky M; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C; Wiley, H Steven; Ahring, Birgitte K; Linggi, Bryan

    2013-01-01

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions.

  14. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    PubMed

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases.

  15. Systems Pharmacology-Based Discovery of Natural Products for Precision Oncology Through Targeting Cancer Mutated Genes.

    PubMed

    Fang, J; Cai, C; Wang, Q; Lin, P; Zhao, Z; Cheng, F

    2017-03-01

    Massive cancer genomics data have facilitated the rapid revolution of a novel oncology drug discovery paradigm through targeting clinically relevant driver genes or mutations for the development of precision oncology. Natural products with polypharmacological profiles have been demonstrated as promising agents for the development of novel cancer therapies. In this study, we developed an integrated systems pharmacology framework that facilitated identifying potential natural products that target mutated genes across 15 cancer types or subtypes in the realm of precision medicine. High performance was achieved for our systems pharmacology framework. In case studies, we computationally identified novel anticancer indications for several US Food and Drug Administration-approved or clinically investigational natural products (e.g., resveratrol, quercetin, genistein, and fisetin) through targeting significantly mutated genes in multiple cancer types. In summary, this study provides a powerful tool for the development of molecularly targeted cancer therapies through targeting the clinically actionable alterations by exploiting the systems pharmacology of natural products.

  16. Systems Pharmacology‐Based Discovery of Natural Products for Precision Oncology Through Targeting Cancer Mutated Genes

    PubMed Central

    Fang, J; Cai, C; Wang, Q; Lin, P

    2017-01-01

    Massive cancer genomics data have facilitated the rapid revolution of a novel oncology drug discovery paradigm through targeting clinically relevant driver genes or mutations for the development of precision oncology. Natural products with polypharmacological profiles have been demonstrated as promising agents for the development of novel cancer therapies. In this study, we developed an integrated systems pharmacology framework that facilitated identifying potential natural products that target mutated genes across 15 cancer types or subtypes in the realm of precision medicine. High performance was achieved for our systems pharmacology framework. In case studies, we computationally identified novel anticancer indications for several US Food and Drug Administration‐approved or clinically investigational natural products (e.g., resveratrol, quercetin, genistein, and fisetin) through targeting significantly mutated genes in multiple cancer types. In summary, this study provides a powerful tool for the development of molecularly targeted cancer therapies through targeting the clinically actionable alterations by exploiting the systems pharmacology of natural products. PMID:28294568

  17. The paf gene product modulates asexual development in Penicillium chrysogenum.

    PubMed

    Hegedüs, Nikoletta; Sigl, Claudia; Zadra, Ivo; Pócsi, Istvan; Marx, Florentine

    2011-06-01

    Penicillium chrysogenum secretes a low molecular weight, cationic and cysteine-rich protein (PAF). It has growth inhibitory activity against the model organism Aspergillus nidulans and numerous zoo- and phytopathogenic fungi but shows only minimal conditional antifungal activity against the producing organism itself. In this study we provide evidence for an additional function of PAF which is distinct from the antifungal activity against putative ecologically concurrent microorganisms. Our data indicate that PAF enhances conidiation in P. chrysogenum by modulating the expression of brlA, the central regulatory gene for mitospore development. A paf deletion strain showed a significant impairment of mitospore formation which sustains our hypothesis that PAF plays an important role in balancing asexual differentiation in P. chrysogenum.

  18. Alphavirus vectors for vaccine production and gene therapy.

    PubMed

    Lundstrom, Kenneth

    2003-06-01

    Alphavirus vectors demonstrate high expression of heterologous proteins in a broad range of host cells. Replication-deficient as well as replication-competent variants exist. Systemic delivery of many viral antigens has elicited strong antibody responses in immunized mice and primates, and protection against challenges with lethal viruses was obtained. Similarly, prophylactic vaccination was established against tumor challenges. Attention has been paid to the engineering of improved targeting to immunologically active cells, such as dendritic cells. In the area of gene therapy, intratumoral injections of alphavirus vectors have resulted in potentially promising tumor rejection. Moreover, encapsulation of alphavirus particles into liposomes demonstrated efficient tumor targeting in mice with severe combined immunodeficiency, which permitted the initiation of clinical trials for patients with advanced kidney carcinoma and melanoma.

  19. Detection of rice tungro bacilliform virus gene products in vivo.

    PubMed

    Hay, J; Grieco, F; Druka, A; Pinner, M; Lee, S C; Hull, R

    1994-12-01

    To study the products of the open reading frames (ORFs) of rice tungro bacilliform virus in rice plants the sequences containing ORFs I (encoding a 24-kDa protein, P24) and IV (P46) and the protease and polymerase (reverse transcriptase+RNaseH) domains of ORF III were cloned into a pGEX expression vector. The proteins, which were C-terminal fusions to glutathione S-transferase, were expressed in Escherichia coli and antisera were raised against them which, together with an antiserum against virus particles, was used to probe blots of proteins from infected and uninoculated plants and from virus preparations. The P24 antiserum detected virus-specific proteins of 74, 60, and 52 kDa, which are much bigger than expected. These proteins were found in virus preparations and immunogold labeling suggested that they might be internal in the particles. Virus-specific proteins of 33, 37, 62, and > 150 kDa were revealed by antiserum to virus particles. The antiserum to the protease revealed proteins of 13.5, 37, and 68 kDa both in extracts from infected plants and in purified virus preparations. This antiserum decorated intact virus particles as did the particle antiserum. The polymerase domain antiserum reacted with products of 56, 65, and 68 kDa in extracts from infected plants but not in virus particles. The antiserum to the ORF IV product did not detect any bands in either infected plant extracts or virus preparations. The significance of these products is discussed.

  20. Cerebrospinal Fluid Levels of Leptin, Proopiomelanocortin, and Agouti-Related Protein in Human Pregnancy: Evidence for Leptin Resistance

    PubMed Central

    Page-Wilson, Gabrielle; Reitman-Ivashkov, Elena; Meece, Kana; White, Anne; Rosenbaum, Michael; Smiley, Richard M.

    2013-01-01

    Context: Leptin suppresses appetite by modulating the expression of hypothalamic neuropeptides including proopiomelanocortin (POMC) and agouti-related peptide (AgRP). Yet during pregnancy, caloric consumption increases despite elevated plasma leptin levels. Design and Participants: To investigate this paradox, we measured leptin and soluble leptin receptor in plasma and leptin, POMC, and AgRP in cerebrospinal fluid (CSF) from 21 fasting pregnant women before delivery by cesarean section at a university hospital and from 14 fasting nonpregnant women. Results: Prepregnancy body mass index was 24.6 ± 1.1 (se) vs. 31.3 ± 1.3 at term vs. 26.5 ± 1.6 kg/m2 in controls. Plasma leptin (32.9 ± 4.6 vs. 16.7 ± 3.0 ng/ml) and soluble leptin receptor (30.9 ± 2.3 vs. 22.1 ± 1.4 ng/ml) levels were significantly higher in pregnant women. However, mean CSF leptin did not differ between the two groups (283 ± 34 vs. 311 ± 32 pg/ml), consistent with a relative decrease in leptin transport into CSF during pregnancy. Accordingly, the CSF/plasma leptin percentage was 1.0 ± 0.01% in pregnant subjects vs. 2.1 ± 0.2% in controls (P < 0.0001). Mean CSF AgRP was significantly higher in pregnant subjects (32.3 ± 2.7 vs. 23.5 ± 2.5 pg/ml; P = 0.03). Mean CSF POMC was not significantly different in pregnant subjects (200 ± 13.6 vs. 229 ± 17.3 fmol/ml; P = 0.190). However, the mean AgRP/POMC ratio was significantly higher among pregnant women (P = 0.003), consistent with an overall decrease in melanocortin tone favoring increased food intake during pregnancy. Conclusions: These data demonstrate that despite peripheral hyperleptinemia, positive energy balance is achieved during pregnancy by a relative decrease in central leptin concentrations and resistance to leptin's effects on target neuropeptides that regulate energy balance. PMID:23118421

  1. The high glycemic index diet was an independent predictor to explain changes in agouti-related protein in obese adolescents.

    PubMed

    Dal Molin Netto, Bárbara; Landi Masquio, Deborah Cristina; Da Silveira Campos, Raquel Munhoz; De Lima Sanches, Priscila; Campos Corgosinho, Flavia; Tock, Lian; Missae Oyama, Lila; Túlio de Mello, Marco; Tufik, Sergio; Dâmaso, Ana Raimunda

    2014-02-01

    La Dieta de alto índice glucémico es un predictor independiente para explicar los cambios en la proteína relacionada al agouti en adolescentes obesos. Introducción y objetivos: El papel de la dieta de índice glucémico (GI) en el control de los factores orexigénicos y anorexígenos del balance de energía todavía no está claro. El presente estudio tuvo como objetivo evaluar si la dieta habitual, de acuerdo con diferentes alimentos con IG, ejerce influencia sobre la regulación de los marcadores del balance de energía y los efectos de la intervención interdisciplinaria en adolescentes obesos. Métodos: Un total de 55 adolescentes obesos, con edades de 14 a 19 años, han sido sometidos a un año de tratamiento interdisciplinario y se dividieron en dos grupos, de acuerdo al patrón de dieta predominante de la ingesta de alimentos: el grupo IG alto (H-GI; n = 29) y GI moderada/bajo grupo (M/L-GI, n = 26). Resultados: La concentración de orexigenic factor de AgRP (p < 0,01), la grasa visceral (p = 0,04) y la relación visceral/ subcutánea (p = 0,03) fueron mayores en el grupo de HGI en comparación con el grupo M/L-GI. Por otra parte, el consumo habitual de alimentos H-GI fue un predictor independiente para explicar los cambios en las concentraciones de AgRP. Después de un año de tratamiento interdisciplinario, los adolescentes presentan una reducción significativa en el peso corporal, la grasa corporal total (%), visceral y la grasa subcutánea y el HOMA-IR, así como un aumento significativo de la masa libre de grasa (%). Conclusiones: Nuestros resultados pueden sugerir que la dieta H-GI habitual podría upregulate vías orexigénicos, contribuyendo al círculo vicioso entre las dietas indeseables, desregula el equilibrio energético y predisponen a la obesidad. Uno por otro lado, un año de tratamiento interdisciplinario puede perfil metabólico mejora significativa y la obesidad central en los adolescentes.

  2. Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

    PubMed

    Moriya, Yuki; Yamada, Takuji; Okuda, Shujiro; Nakagawa, Zenichi; Kotera, Masaaki; Tokimatsu, Toshiaki; Kanehisa, Minoru; Goto, Susumu

    2016-03-28

    Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes.

  3. Physiological evaluation of the filamentous fungus Trichoderma reesei in production processes by marker gene expression analysis

    PubMed Central

    Rautio, Jari J; Bailey, Michael; Kivioja, Teemu; Söderlund, Hans; Penttilä, Merja; Saloheimo, Markku

    2007-01-01

    Background Biologically relevant molecular markers can be used in evaluation of the physiological state of an organism in biotechnical processes. We monitored at high frequency the expression of 34 marker genes in batch, fed-batch and continuous cultures of the filamentous fungus Trichoderma reesei by the transcriptional analysis method TRAC (TRanscript analysis with the aid of Affinity Capture). Expression of specific genes was normalised either with respect to biomass or to overall polyA RNA concentration. Expressional variation of the genes involved in various process relevant cellular functions, such as protein production, growth and stress responses, was related to process parameters such as specific growth and production rates and substrate and dissolved oxygen concentrations. Results Gene expression of secreted cellulases and recombinant Melanocarpus albomyces laccase predicted the trends in the corresponding extracellular enzyme production rates and was highest in a narrow "physiological window" in the specific growth rate (μ) range of 0.03 – 0.05 h-1. Expression of ribosomal protein mRNAs was consistent with the changes in μ. Nine starvation-related genes were found as potential markers for detection of insufficient substrate feed for maintaining optimal protein production. For two genes induced in anaerobic conditions, increasing transcript levels were measured as dissolved oxygen decreased. Conclusion The data obtained by TRAC supported the usefulness of focused and intensive transcriptional analysis in monitoring of biotechnical processes providing thus tools for process optimisation purposes. PMID:17537269

  4. Regulation of a novel gene cluster involved in secondary metabolite production in Streptomyces coelicolor.

    PubMed

    Hindra; Pak, Patricia; Elliot, Marie A

    2010-10-01

    Antibiotic biosynthesis in the streptomycetes is a complex and highly regulated process. Here, we provide evidence for the contribution of a novel genetic locus to antibiotic production in Streptomyces coelicolor. The overexpression of a gene cluster comprising four protein-encoding genes (abeABCD) and an antisense RNA-encoding gene (α-abeA) stimulated the production of the blue-pigmented metabolite actinorhodin on solid medium. Actinorhodin production also was enhanced by the overexpression of an adjacent gene (abeR) encoding a predicted Streptomyces antibiotic regulatory protein (SARP), while the deletion of this gene impaired actinorhodin production. We found the abe genes to be differentially regulated and controlled at multiple levels. Upstream of abeA was a promoter that directed the transcription of abeABCD at a low but constitutive level. The expression of abeBCD was, however, significantly upregulated at a time that coincided with the initiation of aerial development and the onset of secondary metabolism; this expression was activated by the binding of AbeR to four heptameric repeats upstream of a promoter within abeA. Expressed divergently to the abeBCD promoter was α-abeA, whose expression mirrored that of abeBCD but did not require activation by AbeR. Instead, α-abeA transcript levels were subject to negative control by the double-strand-specific RNase, RNase III.

  5. Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence

    PubMed Central

    Bauer, Beth A.; Stevens, Marla K.; Hansen, Eric J.

    1998-01-01

    The lipooligosaccharide (LOS) present in the outer membrane of Haemophilus ducreyi is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that had 87% identity with the protein product of the gmhA (isn) gene of Haemophilus influenzae. In H. influenzae type b, inactivation of the gmhA gene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396–402, 1996). The H. ducreyi gmhA gene was able to complement a gmhA-deficient Escherichia coli strain, a result which confirmed the identity of this gene. When the gmhA gene of H. ducreyi was inactivated by insertion of a cat cartridge, the resultant H. ducreyi gmhA mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type H. ducreyi strain and its isogenic gmhA mutant were used in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi, the gmhA mutant was found to be substantially less virulent than the wild-type parent strain. The H. ducreyi gmhA gene was amplified by PCR from the H. ducreyi chromosome and cloned into the pLS88 vector. When the H. ducreyi gmhA gene was present in trans in gmhA mutant 35000.252, expression of the gmhA gene product restored the virulence of this mutant to wild-type levels. These results indicate that the gmhA gene product of H. ducreyi is essential for the expression of wild-type LOS by this pathogen. PMID:9712780

  6. Phylogenomic study of lipid genes involved in microalgal biofuel production-candidate gene mining and metabolic pathway analyses.

    PubMed

    Misra, Namrata; Panda, Prasanna Kumar; Parida, Bikram Kumar; Mishra, Barada Kanta

    2012-01-01

    Optimizing microalgal biofuel production using metabolic engineering tools requires an in-depth understanding of the structure-function relationship of genes involved in lipid biosynthetic pathway. In the present study, genome-wide identification and characterization of 398 putative genes involved in lipid biosynthesis in Arabidopsis thaliana Chlamydomonas reinhardtii, Volvox carteri, Ostreococcus lucimarinus, Ostreococcus tauri and Cyanidioschyzon merolae was undertaken on the basis of their conserved motif/domain organization and phylogenetic profile. The results indicated that the core lipid metabolic pathways in all the species are carried out by a comparable number of orthologous proteins. Although the fundamental gene organizations were observed to be invariantly conserved between microalgae and Arabidopsis genome, with increased order of genome complexity there seems to be an association with more number of genes involved in triacylglycerol (TAG) biosynthesis and catabolism. Further, phylogenomic analysis of the genes provided insights into the molecular evolution of lipid biosynthetic pathway in microalgae and confirm the close evolutionary proximity between the Streptophyte and Chlorophyte lineages. Together, these studies will improve our understanding of the global lipid metabolic pathway and contribute to the engineering of regulatory networks of algal strains for higher accumulation of oil.

  7. Split-gene system for hybrid wheat seed production

    PubMed Central

    Kempe, Katja; Rubtsova, Myroslava; Gils, Mario

    2014-01-01

    Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility. The barnase coding information is divided and distributed at two loci that are located on allelic positions of the host chromosome and are therefore “linked in repulsion.” Functional complementation of the loci is achieved through coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragments. This system allows for growth and maintenance of male-sterile female crossing partners, whereas the hybrids are fertile. The technology does not require fertility restorers and is based solely on the genetic modification of the female crossing partner. PMID:24821800

  8. Split-gene system for hybrid wheat seed production.

    PubMed

    Kempe, Katja; Rubtsova, Myroslava; Gils, Mario

    2014-06-24

    Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility. The barnase coding information is divided and distributed at two loci that are located on allelic positions of the host chromosome and are therefore "linked in repulsion." Functional complementation of the loci is achieved through coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragments. This system allows for growth and maintenance of male-sterile female crossing partners, whereas the hybrids are fertile. The technology does not require fertility restorers and is based solely on the genetic modification of the female crossing partner.

  9. A mutant gene that increases gibberellin production in Brassica

    SciTech Connect

    Rood, S.B. ); Williams, P.H. ); Pearce, D.; Pharis, R.P. ); Murofushi, Noboru ); Mander, L.N. )

    1990-07-01

    A single gene mutant (elongated internode (ein/ein)) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A{sub 3} (GA{sub 3}) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA{sub 1} and GA{sub 3} were estimated by gas chromatography-selected ion monitoring using ({sup 2}H)GA{sub 1} as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA{sub 20} and GA{sub 1}, and the rate of GA{sub 19} metabolism were simultaneously analyzed. Levels of GA{sub 1} and GA{sub 20} were 4.6- and 12.9-fold higher, respectively, and conversions to GA{sub 20} and GA{sub 1} were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA{sub 1} biosynthesis in ein, the conversion of ({sup 3}H)GA{sub 20} to ({sup 3}H) GA{sub 1} was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA{sub 1} biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A{sub 1} and A{sub 3}.

  10. Expanded natural product diversity revealed by analysis of lanthipeptide-like gene clusters in actinobacteria.

    PubMed

    Zhang, Qi; Doroghazi, James R; Zhao, Xiling; Walker, Mark C; van der Donk, Wilfred A

    2015-07-01

    Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Lanthipeptides are widely distributed in taxonomically distant species, and their currently known biosynthetic systems and biological activities are diverse. Building on the recent natural product gene cluster family (GCF) project, we report here large-scale analysis of lanthipeptide-like biosynthetic gene clusters from Actinobacteria. Our analysis suggests that lanthipeptide biosynthetic pathways, and by extrapolation the natural products themselves, are much more diverse than currently appreciated and contain many different posttranslational modifications. Furthermore, lanthionine synthetases are much more diverse in sequence and domain topology than currently characterized systems, and they are used by the biosynthetic machineries for natural products other than lanthipeptides. The gene cluster families described here significantly expand the chemical diversity and biosynthetic repertoire of lanthionine-related natural products. Biosynthesis of these novel natural products likely involves unusual and unprecedented biochemistries, as illustrated by several examples discussed in this study. In addition, class IV lanthipeptide gene clusters are shown not to be silent, setting the stage to investigate their biological activities.

  11. Expanded Natural Product Diversity Revealed by Analysis of Lanthipeptide-Like Gene Clusters in Actinobacteria

    PubMed Central

    Zhang, Qi; Doroghazi, James R.; Zhao, Xiling; Walker, Mark C.

    2015-01-01

    Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Lanthipeptides are widely distributed in taxonomically distant species, and their currently known biosynthetic systems and biological activities are diverse. Building on the recent natural product gene cluster family (GCF) project, we report here large-scale analysis of lanthipeptide-like biosynthetic gene clusters from Actinobacteria. Our analysis suggests that lanthipeptide biosynthetic pathways, and by extrapolation the natural products themselves, are much more diverse than currently appreciated and contain many different posttranslational modifications. Furthermore, lanthionine synthetases are much more diverse in sequence and domain topology than currently characterized systems, and they are used by the biosynthetic machineries for natural products other than lanthipeptides. The gene cluster families described here significantly expand the chemical diversity and biosynthetic repertoire of lanthionine-related natural products. Biosynthesis of these novel natural products likely involves unusual and unprecedented biochemistries, as illustrated by several examples discussed in this study. In addition, class IV lanthipeptide gene clusters are shown not to be silent, setting the stage to investigate their biological activities. PMID:25888176

  12. Expression of MAGE-1 and -3 genes and gene products in human hepatocellular carcinoma

    PubMed Central

    Kariyama, K; Higashi, T; Kobayashi, Y; Nouso, K; Nakatsukasa, H; Yamano, T; Ishizaki, M; Kaneyoshi, T; Toshikuni, N; Ohnishi, T; Fujiwara, K; Nakayama, E; Terracciano, L; Spagnoli, G C; Tsuji, T

    1999-01-01

    MAGE gene family encodes peptides recognized by autologous cytotoxic T lymphocytes in a major histocompatibility complex (MHC) class-I restricted fashion. In the present study, we have performed reverse-transcription polymerase chain reaction (RT-PCR) for the genes, as well as immunohistochemical analysis and Western blotting of MAGE-1 and -3 proteins in 33 surgically resected hepatocellular carcinomas (HCCs). MAGE-1 and -3 mRNAs were constitutively expressed exclusively in 78 and 42% of HCCs respectively. On immunohistochemistry with monoclonal antibodies, 77B for MAGE-1 and 57B for MAGE-3, MAGE-1 and -3 proteins were recognized in cytoplasm of only six among 33 (18%) and two of 29 HCCs (7%) respectively. The distribution pattern was mostly focal in HCC nodules. By contrast, the Western blot analysis revealed that the MAGE-1 (46 kDa) and -3 proteins (48 kDa) were expressed in 80 and 60% of 15 HCCs examined respectively. The proteins of MAGE-1 and -3 were also expressed exclusively in HCCs regardless of the histological grading and clinical staging. Our results indicate that the detection of the genes by RT-PCR or the proteins by Western blotting is useful for differentiating early HCCs from non-cancerous lesions, and that the peptides derived from MAGE-1 and -3 proteins might be suitable targets for immunotherapy of human HCC. © 1999 Cancer Research Campaign PMID:10576668

  13. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  14. Genetic resources for advanced biofuel production described with the Gene Ontology

    PubMed Central

    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, João C.; Mukhopadhyay, Biswarup; Tyler, Brett M.

    2014-01-01

    Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary. The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology () project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. Here we review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way. PMID:25346727

  15. Genetic resources for advanced biofuel production described with the Gene Ontology

    SciTech Connect

    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, Joao C.; Mukhopadhyay, Biswarup; Tyler, Brett M.

    2014-10-10

    Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary.The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology (http://www.mengo.biochem.vt.edu) project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. We review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.

  16. Genetic resources for advanced biofuel production described with the Gene Ontology

    DOE PAGES

    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; ...

    2014-10-10

    Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary.The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology (http://www.mengo.biochem.vt.edu) project is extending the GO to include new terms to describe microbial processes of interest to bioenergymore » production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. We review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.« less

  17. Products of lipid, protein and RNA oxidation as signals and regulators of gene expression in plants

    PubMed Central

    Chmielowska-Bąk, Jagna; Izbiańska, Karolina; Deckert, Joanna

    2015-01-01

    Reactive oxygen species (ROS) are engaged in several processes essential for normal cell functioning, such as differentiation, anti-microbial defense, stimulus sensing and signaling. Interestingly, recent studies imply that cellular signal transduction and gene regulation are mediated not only directly by ROS but also by the molecules derived from ROS-mediated oxidation. Lipid peroxidation leads to non-enzymatic formation of oxylipins. These molecules were shown to modulate expression of signaling associated genes including genes encoding phosphatases, kinases and transcription factors. Oxidized peptides derived from protein oxidation might be engaged in organelle-specific ROS signaling. In turn, oxidation of particular mRNAs leads to decrease in the level of encoded proteins and thus, contributes to the post-transcriptional regulation of gene expression. Present mini review summarizes latest findings concerning involvement of products of lipid, protein and RNA oxidation in signal transduction and gene regulation. PMID:26082792

  18. ROS production during symbiotic infection suppresses pathogenesis-related gene expression.

    PubMed

    Peleg-Grossman, Smadar; Melamed-Book, Naomi; Levine, Alex

    2012-03-01

    Leguminous plants have exclusive ability to form symbiotic relationship with soil bacteria of the genus Rhizobium. Symbiosis is a complex process that involves multiple molecular signaling activities, such as calcium fluxes, production of reactive oxygen species (ROS) and synthesis of nodulation genes. We analyzed the role of ROS in defense gene expression in Medicago truncatula during symbiosis and pathogenesis. Studies in Arabidopsis thaliana showed that the induction of pathogenesis-related (PR) genes during systemic acquired resistance (SAR) is regulated by NPR1 protein, which resides in the cytoplasm as an oligomer. After oxidative burst and return of reducing conditions, the NPR1 undergoes monomerization and becomes translocated to the nucleus, where it functions in PR genes induction. We show that ROS production is both stronger and longer during symbiotic interactions than during interactions with pathogenic, nonhost or common nonpathogenic soil bacteria. Moreover, root cells inoculated with Sinorhizobium meliloti accumulated ROS in the cytosol but not in vacuoles, as opposed to Pseudomonas putida inoculation or salt stress treatment. Furthermore, increased ROS accumulation by addition of H₂O₂ reduced the PR gene expression, while catalase had an opposite effect, establishing that the PR gene expression is opposite to the level of cytoplasmic ROS. In addition, we show that salicylic acid pretreatment significantly reduced ROS production in root cells during symbiotic interaction.

  19. Associations between polymorphisms of the gene and milk production traits in water buffaloes.

    PubMed

    Deng, T X; Pang, C Y; Lu, X R; Zhu, P; Duan, A Q; Liang, X W

    2016-03-01

    Signal transducer and activator of transcription 1 () is an important regulator of mammary gland differentiation and cell survival that has been regarded as a candidate gene affecting milk production traits in mammals. Therefore, this study was conducted to evaluate significant associations between SNP of the gene and milk production traits in buffaloes. Here, 18 SNP were identified in the buffalo gene, including 15 intronic mutations and 3 exon mutations. All the identified SNP were then genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry methods from 192 buffaloes. All the SNP were in Hardy-Weinberg equilibrium, and 2 haplotype blocks were successfully constructed based on these SNP data, which formed 5 and 3 major haplotypes in the population (>5%), respectively. The results of association analysis showed that only SNP13 located in exon 10 was significantly associated with the milk production traits in the population ( < 0.05). Single nucleotide polymorphism 2, SNP5, SNP8, and SNP9 were associated with protein percentage, and SNP4 and SNP10 were associated with 305-d milk yield ( < 0.05). Our results provide evidence that polymorphisms of the buffalo gene are associated with milk production traits and can be used as a candidate gene for marker-assisted selection in buffalo breeding.

  20. Engineering validamycin production by tandem deletion of γ-butyrolactone receptor genes in Streptomyces hygroscopicus 5008.

    PubMed

    Tan, Gao-Yi; Peng, Yao; Lu, Chenyang; Bai, Linquan; Zhong, Jian-Jiang

    2015-03-01

    Paired homologs of γ-butyrolactone (GBL) biosynthesis gene afsA and GBL receptor gene arpA are located at different positions in genome of Streptomyces hygroscopicus 5008. Inactivation of afsA homologs dramatically decreased biosynthesis of validamycin, an important anti-fungal antibiotic and a critical substrate for antidiabetic drug synthesis, and the deletion of arpA homologs increased validamycin production by 26% (ΔshbR1) and 20% (ΔshbR3). By double deletion, the ΔshbR1/R3 mutant showed higher transcriptional levels of adpA-H (the S. hygroscopicus ortholog of the global regulatory gene adpA) and validamycin biosynthetic genes, and validamycin production increased by 55%. Furthermore, by engineering a high-producing industrial strain via tandem deletion of GBL receptor genes, validamycin production and productivity were enhanced from 19 to 24 g/L (by 26%) and from 6.7 to 9.7 g/L(-1) d(-1) (by 45%), respectively, which was the highest ever reported. The strategy demonstrated here may be useful to engineering other Streptomyces spp. with multiple pairs of afsA-arpA homologs.

  1. The product of the imprinted H19 gene is an oncofetal RNA.

    PubMed Central

    Ariel, I.; Ayesh, S.; Perlman, E. J.; Pizov, G.; Tanos, V.; Schneider, T.; Erdmann, V. A.; Podeh, D.; Komitowski, D.; Quasem, A. S.; de Groot, N.; Hochberg, A.

    1997-01-01

    AIMS/BACKGROUND: The H19 gene is an imprinted, maternally expressed gene in humans. It is tightly linked and coregulated with the imprinted, paternally expressed gene of insulin-like growth factor 2. The H19 gene product is not translated into protein and functions as an RNA molecule. Although its role has been investigated for more than a decade, its biological function is still not understood fully. H19 is abundantly expressed in many tissues from early stages of embryogenesis through fetal life, and is down regulated postnatally. It is also expressed in certain childhood and adult tumours. This study was designed to screen the expression of H19 in human cancer and its relation to the expression of H19 in the fetus. METHODS: Using in situ hybridisation with a [35S] labelled probe, H19 mRNA was detected in paraffin wax sections of fetal tissues from the first and second trimesters of pregnancy and of a large array of human adult and childhood tumours arising from these tissues. RESULTS: The H19 gene is expressed in tumours arising from tissues which express this gene in fetal life. Its expression in the fetus and in cancer is closely linked with tissue differentiation. CONCLUSIONS: Based on these and previous data, H19 is neither a tumour suppressor gene nor an oncogene. Its product is an oncofetal RNA. The potential use of this RNA as a tumour marker should be evaluated. Images PMID:9208812

  2. Investigation of genes involved in nisin production in Enterococcus spp. strains isolated from raw goat milk.

    PubMed

    Perin, Luana Martins; Todorov, Svetoslav Dimitrov; Nero, Luís Augusto

    2016-09-01

    Different strains of Lactococcus lactis are capable of producing the bacteriocin nisin. However, genetic transfer mechanisms allow the natural occurrence of genes involved in nisin production in members of other bacterial genera, such as Enterococcus spp. In a previous study, nisA was identified in eight enterococci capable of producing antimicrobial substances. The aim of this study was to verify the presence of genes involved in nisin production in Enterococcus spp. strains, as well as nisin expression. The nisA genes from eight Enterococcus spp. strains were sequenced and the translated amino acid sequences were compared to nisin amino-acid sequences previously described in databases. Although containing nisin structural and maturation related genes, the enterococci strains tested in the present study did not present the immunity related genes (nisFEG and nisI). The translated sequences of nisA showed some point mutations, identical to those presented by Lactococcus strains isolated from goat milk. All enterococci were inhibited by nisin, indicating the absence of immunity and thus that nisin cannot be expressed. This study demonstrated for the first time the natural occurrence of nisin structural genes in Enterococcus strains and highlights the importance of providing evidence of a link between the presence of bacteriocin genes and their expression.

  3. Overexpression of bacterial ethylene-forming enzyme gene in Trichoderma reesei enhanced the production of ethylene

    PubMed Central

    Chen, Xi; Liang, Yong; Hua, Jing; Tao, Li; Qin, Wensheng; Chen, Sanfeng

    2010-01-01

    In order to efficiently utilize natural cellulose materials to produce ethylene, three expression vectors containing the ethylene-forming enzyme (efe) gene from Pseudomonas syringae pv. glycinea were constructed. The target gene was respectively controlled by different promoters: cbh I promoter from Trichoderma reesei cellobiohydrolases I gene, gpd promoter from Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene and pgk I promoter from T. reesei 3-phosphoglycerate kinase I gene. After transforming into T. reesei QM9414, 43 stable transformants were obtained by PCR amplification and ethylene determination. Southern blot analysis of 14 transformants demonstrated that the efe gene was integrated into chromosomal DNA with copy numbers from 1 to 4. Reverse transcription polymerase chain reaction (RT-PCR) analysis of 6 transformants showed that the heterologous gene was transcribed. By using wheat straw as a carbon source, the ethylene production rates of aforementioned 14 transformants were measured. Transformant C30-3 with pgk I promoter had the highest ethylene production (4,012 nl h-1 l-1). This indicates that agricultural wastes could be used to produce ethylene in recombinant filamentous fungus T. reesei. PMID:20150979

  4. New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters

    PubMed Central

    Luo, Yunzi; Enghiad, Behnam; Zhao, Huimin

    2015-01-01

    Natural product scaffolds remain a major source and inspiration for human therapeutics. However, generation of a natural product in the post-genomic era often requires reconstruction of the corresponding biosynthetic gene cluster in a heterologous host. In the burgeoning fields of synthetic biology and metabolic engineering, a significant amount of efforts has been devoted to develop DNA assembly techniques with higher efficiency, fidelity, and modularity, and heterologous expression systems with higher productivity and yield. Here we describe recent advances in DNA assembly and host engineering and highlight their applications in natural product discovery and engineering. PMID:26647833

  5. New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters.

    PubMed

    Luo, Yunzi; Enghiad, Behnam; Zhao, Huimin

    2016-02-01

    Natural product scaffolds remain a major source and inspiration for human therapeutics. However, generation of a natural product in the post-genomic era often requires reconstruction of the corresponding biosynthetic gene cluster in a heterologous host. In the burgeoning fields of synthetic biology and metabolic engineering, a significant amount of efforts has been devoted to develop DNA assembly techniques with higher efficiency, fidelity, and modularity, and heterologous expression systems with higher productivity and yield. Here we describe recent advances in DNA assembly and host engineering and highlight their applications in natural product discovery and engineering.

  6. Production of indole antibiotics induced by exogenous gene derived from sponge metagenomes.

    PubMed

    Takeshige, Yuya; Egami, Yoko; Wakimoto, Toshiyuki; Abe, Ikuro

    2015-05-01

    Sponge metagenomes are accessible genetic sources containing genes and gene clusters responsible for the biosynthesis of sponge-derived bioactive natural products. In this study, we obtained the clone pDC112, producing turbomycin A and 2,2-di(3-indolyl)-3-indolone, based on the functional screening of the metagenome library derived from the marine sponge Discodermia calyx. The subcloning experiment identified ORF 25, which is homologous to inosine 5'-monophosphate dehydrogenase and required for the production of 2,2-di(3-indolyl)-3-indolone in Escherichia coli.

  7. Analysis of the genetic effects of prolactin gene polymorphisms on chicken egg production.

    PubMed

    Li, Hui-Fang; Shu, Jing-Ting; Du, Yu-Feng; Shan, Yan-Ju; Chen, Kuan-Wei; Zhang, Xue-Yu; Han, Wei; Xu, Wen-Juan

    2013-01-01

    Chicken prolactin (PRL) is a physiological candidate gene for egg production. Variations of T8052C and G8113C in exon 5 of PRL gene may associate with chicken egg production. The objective of the study was to investigate the association of these two single nucleotide polymorphisms in PRL gene with egg production of Recessive White chickens and Qingyuan Partridge chickens. Genotyping was performed by polymerase chain reaction-ligase detection reaction (PCR-LDR) method. The T8052C and G8113C of PRL were significantly associated with age at first egg (AFE) and total egg number at 300 days of age (EN 300). A significant association was also found between T8052C-G8113C haplotypes and AFE as well as EN300, the H2H3 was the most advantageous diplotype for egg production. We putatively drew the conclusion that these two SNPs in PRL gene as well as their haplotypes could be used as the potential molecular markers for egg production traits in chicken.

  8. Biofilm formation, phenotypic production of cellulose and gene expression in Salmonella enterica decrease under anaerobic conditions.

    PubMed

    Lamas, A; Miranda, J M; Vázquez, B; Cepeda, A; Franco, C M

    2016-12-05

    Salmonella enterica subsp. enterica is one of the main food-borne pathogens. This microorganism combines an aerobic life outside the host with an anaerobic life within the host. One of the main concerns related to S. enterica is biofilm formation and cellulose production. In this study, biofilm formation, morphotype, cellulose production and transcription of biofilm and quorum sensing-related genes of 11 S. enterica strains were tested under three different conditions: aerobiosis, microaerobiosis, and anaerobiosis. The results showed an influence of oxygen levels on biofilm production. Biofilm formation was significantly higher (P<0.05) in aerobiosis than in microaerobiosis and anaerobiosis. Cellulose production and RDAR (red, dry, and rough) were expressed only in aerobiosis. In microaerobiosis, the strains expressed the SAW (smooth and white) morphotype, while in anaerobiosis the colonies appeared small and red. The expression of genes involved in cellulose synthesis (csgD and adrA) and quorum sensing (sdiA and luxS) was reduced in microaerobiosis and anaerobiosis in all S. enterica strains tested. This gene expression levels were less reduced in S. Typhimurium and S. Enteritidis compared to the tested serotypes. There was a relationship between the expression of biofilm and quorum sensing-related genes. Thus, the results from this study indicate that biofilm formation and cellulose production are highly influenced by atmospheric conditions. This must be taken into account as contamination with these bacteria can occur during food processing under vacuum or modified atmospheres.

  9. Genetic variation within coat color genes of MC1R and ASIP in Chinese brownish red Tibetan pigs.

    PubMed

    Mao, Huirong; Ren, Jun; Ding, Nengshui; Xiao, Shijun; Huang, Lusheng

    2010-12-01

    Melanocortin receptor 1 (MC1R) and agouti signaling protein (ASIP) are two major genes affecting coat color phenotypes in mammals, and inactivation mutations in the MC1R gene are responsible for red coat color in European pig breeds. Conversely, the gain-of-function ASIP mutations block MC1R signaling and lead to the production of red pheomelanin. Chinese Tibetan pigs have three types of coat color phenotypes, including brownish red, solid black and black with patches of brownish red and white. Herein, we investigated variations of the MC1R and ASIP genes in Tibetan pigs. The results showed that the brownish red Tibet pig had the dominant black MC1R allele (E(D1)). No loss-of-function mutation in MC1R responsible for red coat color in European breeds was observed in this breed. No causal mutation for the red coat color phenotype was found in the coding sequence of the ASIP gene. A novel missense mutation c.157G > A was firstly identified in exon 2 of ASIP, which was further genotyped in 285 pigs from five Chinese breeds and three Western breeds having different coat color phenotypes. Nearly all pigs were GG homozygotes. In conclusion, no functional variant responsible for brownish red coloration was found in the coding region of MC1R and ASIP in Tibetan pigs.

  10. 1,3-Propanediol production by Escherichia coli using genes from Citrobacter freundii atcc 8090.

    PubMed

    Przystałowska, Hanna; Zeyland, Joanna; Kośmider, Alicja; Szalata, Marlena; Słomski, Ryszard; Lipiński, Daniel

    2015-01-01

    Compared with chemical synthesis, fermentation has the advantage of mass production at low cost, and has been used in the production of various industrial chemicals. As a valuable organic compound, 1,3-propanediol (1,3-PDO) has numerous applications in the production of polymers, lubricants, cosmetics and medicines. Here, conversion of glycerol (a renewable substrate and waste from biodiesel production) to 1,3-PDO by E. coli bacterial strain carrying altered glycerol metabolic pathway was investigated. Two gene constructs containing the 1,3-PDO operon from Citrobacter freundii (pCF1 and pCF2) were used to transform the bacteria. The pCF1 gene expression construct contained dhaBCE genes encoding the three subunits of glycerol dehydratase, dhaF encoding the large subunit of the glycerol dehydratase reactivation factor and dhaG encoding the small subunit of the glycerol dehydratase reactivating factor. The pCF2 gene expression construct contained the dhaT gene encoding the 1,3-propanediol dehydrogenase. Expression of the genes cloned in the above constructs was under regulation of the T7lac promoter. RT-PCR, SDS-PAGE analyses and functional tests confirmed that 1,3-PDO synthesis pathway genes were expressed at the RNA and protein levels, and worked flawlessly in the heterologous host. In a batch flask culture, in a short time applied just to identify the 1,3-PDO in a preliminary study, the recombinant E. coli bacteria produced 1.53 g/L of 1,3-PDO, using 21.2 g/L of glycerol in 72 h. In the Sartorius Biostat B Plus reactor, they produced 11.7 g/L of 1,3-PDO using 24.2 g/L of glycerol, attaining an efficiency of 0.58 [mol1,3-PDO/molglycerol].

  11. Diversity of Antibiotic Resistance Genes in Enterococcus Strains Isolated from Ready-to-Eat Meat Products.

    PubMed

    Chajęcka-Wierzchowska, Wioleta; Zadernowska, Anna; Łaniewska-Trokenheim, Łucja

    2016-10-25

    The objective of the study was to answer the question of whether the ready-to-eat meat products can pose indirect hazard for consumer health serving as reservoir of Enterococcus strains harboring tetracyclines, aminoglycosides, and macrolides resistance genes. A total of 390 samples of ready-to-eat meat products were investigated. Enterococcus strains were found in 74.1% of the samples. A total of 302 strains were classified as: Enterococcus faecalis (48.7%), Enterococcus faecium (39.7%), Enterococcus casseliflavus (4.3%), Enterococcus durans (3.0%), Enterococcus hirae (2.6%), and other Enterococcus spp. (1.7%). A high percentage of isolates were resistant to streptomycin high level (45%) followed by erythromycin (42.7%), fosfomycin (27.2%), rifampicin (19.2%), tetracycline (36.4%), tigecycline (19.9%). The ant(6')-Ia gene was the most frequently found gene (79.6%). Among the other genes that encode aminoglycosides-modifying enzymes, the highest portion of the strains had the aac(6')-Ie-aph(2'')-Ia (18.5%) and aph(3'')-IIIa (16.6%), but resistance of isolates from food is also an effect of the presence of aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id genes. Resistance to tetracyclines was associated with the presence of tetM (43.7%), tetL (32.1%), tetK (14.6%), tetW (0.7%), and tetO (0.3%) genes. The ermB and ermA genes were found in 33.8% and 18.9% of isolates, respectively. Nearly half of the isolates contained a conjugative transposon of the Tn916/Tn1545 family. Enterococci are widely present in retail ready-to-eat meat products. Many isolated strains (including such species as E. casseliflavus, E. durans, E. hirae, and Enterococcus gallinarum) are antibiotic resistant and carry transferable resistance genes.

  12. Identification and engineering of regulation-related genes toward improved kasugamycin production.

    PubMed

    Zhu, Chenchen; Kang, Qianjin; Bai, Linquan; Cheng, Lin; Deng, Zixin

    2016-02-01

    Kasugamycin, produced by Streptomyces kasugaensis and Streptomyces microaureus, is an important amino-glycoside family antibiotic and widely used for veterinary and agricultural applications. In the left flanking region of the previously reported kasugamycin gene cluster, four additional genes (two-component system kasW and kasX, MerR-family kasV, and isoprenylcysteine carboxyl methyltransferase kasS) were identified both in the low-yielding S. kasugaensis BCRC12349 and high-yielding S. microaureus XM301. Deletion of regulatory gene kasT abolished kasugamycin production, and its overexpression in BCRC12349 resulted in an increased titer by 186 %. Deletion of kasW, kasX, kasV, and kasS improved kasugamycin production by 12, 19, 194, and 22 %, respectively. qRT-PCR analysis demonstrated that the transcription of kas genes was significantly increased in all the four mutants. Similar gene inactivation was performed in the high-yielding strain S. microaureus XM301. As expected, the deletion of kasW/X resulted in a 58 % increase of the yield from 6 to 9.5 g/L. However, the deletion of kasV and over-expression of kasT had no obvious effect, and the disruption of kasS surprisingly decreased kasugamycin production. In addition, trans-complementation of the kasS mutant with a TTA codon-mutated kasS increased the kasugamycin yield by 20 %. A much higher transcription of kas genes was detected in the high-yielding XM301 than in the low-yielding BCRC12349, which may partially account for the discrepancy of gene inactivation effects between them. Our work not only generated engineered strains with improved kasugamycin yield, but also pointed out that different strategies on manipulating regulatory-related genes should be considered for low-yielding or high-yielding strains.

  13. Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

    DOEpatents

    Dai, Ziyu; Lasure, Linda L.; Magnuson, Jon K.

    2008-11-11

    The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

  14. Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

    DOEpatents

    Dai, Ziyu; Lasure, Linda L; Magnuson, Jon K

    2014-05-27

    The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

  15. Isolated Fungal Promoters and Gene Transcription Terminators and Methods of Protein and Chemical Production in a Fungus

    DOEpatents

    Dai, Ziyu; Lasure, Linda L.; Magnuson, Jon K.

    2008-11-11

    The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

  16. Regulatory structures for gene therapy medicinal products in the European Union.

    PubMed

    Klug, Bettina; Celis, Patrick; Carr, Melanie; Reinhardt, Jens

    2012-01-01

    Taking into account the complexity and technical specificity of advanced therapy medicinal products: (gene and cell therapy medicinal products and tissue engineered products), a dedicated European regulatory framework was needed. Regulation (EC) No. 1394/2007, the "ATMP Regulation" provides tailored regulatory principles for the evaluation and authorization of these innovative medicines. The majority of gene or cell therapy product development is carried out by academia, hospitals, and small- and medium-sized enterprises (SMEs). Thus, acknowledging the particular needs of these types of sponsors, the legislation also provides incentives for product development tailored to them. The European Medicines Agency (EMA) and, in particular, its Committee for Advanced Therapies (CAT) provide a variety of opportunities for early interaction with developers of ATMPs to enable them to have early regulatory and scientific input. An important tool to promote innovation and the development of new medicinal products by micro-, small-, and medium-sized enterprises is the EMA's SME initiative launched in December 2005 to offer financial and administrative assistance to smaller companies. The European legislation also foresees the involvement of stakeholders, such as patient organizations, in the development of new medicines. Considering that gene therapy medicinal products are developed in many cases for treatment of rare diseases often of monogenic origin, the involvement of patient organizations, which focus on rare diseases and genetic and congenital disorders, is fruitful. Two such organizations are represented in the CAT. Research networks play another important role in the development of gene therapy medicinal products. The European Commission is funding such networks through the EU Sixth Framework Program.

  17. High-affinity L-arabinose transport operon. Nucleotide sequence and analysis of gene products.

    PubMed

    Scripture, J B; Voelker, C; Miller, S; O'Donnell, R T; Polgar, L; Rade, J; Horazdovsky, B F; Hogg, R W

    1987-09-05

    The nucleotide sequence of the "high-affinity" L-arabinose transport operon has been determined 3' from the regulatory region and found to contain three open reading frames designated araF, araG and araH. The first gene 3' to the regulatory region, araF, encodes the 23-residue signal peptide and the 306-residue mature form of the L-arabinose binding protein (33,200 Mr). The binding protein, which has been described elsewhere, is hydrophilic, soluble and found in the periplasm of Escherichia coli. This gene is followed by an intragenic space of 72 nucleotides, which contains a region of dyad symmetry 23 nucleotides long capable of forming an 11-member stem-loop. The second gene, designated araG, contains an open reading frame capable of encoding an equally hydrophilic protein containing 504 residues (55,000 Mr). Following a 14-nucleotide spacer, which does not appear to have any secondary structure, the third open reading frame, herein designated araH, is capable of encoding a hydrophobic protein containing 329 residues (34,000 Mr) that can only be envisioned as having an integral membrane location. 3' to araH there is a T-rich region containing a 24-nucleotide area of dyad symmetry centered 55 nucleotides from the termination codon. Analysis of the derived primary sequences of the araG and araH products indicates the nature and potential features of these components. The araG protein was found to possess internal homology between its amino and carboxyl-terminal halves, suggesting a common origin. The araG gene product has been shown to be homologous to the rbsA gene product, the hisP product, the ptsB product and the malK product, all of which presumably play similar roles in their respective transport systems. Putative ATP binding sites are observed within the regions of homology. The araH gene product has been shown to be homologous to the rbsC gene product, which is the first observed homology between two purported membrane proteins.

  18. Development of Ecogenomic Sensors for Remote Detection of Marine Microbes, Their Genes and Gene Products

    NASA Astrophysics Data System (ADS)

    Scholin, C.; Preston, C.; Harris, A.; Birch, J.; Marin, R.; Jensen, S.; Roman, B.; Everlove, C.; Makarewicz, A.; Riot, V.; Hadley, D.; Benett, W.; Dzenitis, J.

    2008-12-01

    An internet search using the phrase "ecogenomic sensor" will return numerous references that speak broadly to the idea of detecting molecular markers indicative of specific organisms, genes or other biomarkers within an environmental context. However, a strict and unified definition of "ecogenomic sensor" is lacking and the phrase may be used for laboratory-based tools and techniques as well as semi or fully autonomous systems that can be deployed outside of laboratory. We are exploring development of an ecogenomic sensor from the perspective of a field-portable device applied towards oceanographic research and water quality monitoring. The device is known as the Environmental Sample Processor, or ESP. The ESP employs wet chemistry molecular analytical techniques to autonomously assess the presence and abundance of specific organisms, their genes and/or metabolites in near real-time. Current detection chemistries rely on low- density DNA probe and protein arrays. This presentation will emphasize results from 2007-8 field trials when the ESP was moored in Monterey Bay, CA, as well as current engineering activities for improving analytical capacity of the instrument. Changes in microbial community structure at the rRNA level were observed remotely in accordance with changing chemical and physical oceanographic conditions. Current developments include incorporation of a reusable solid phase extraction column for purifying nucleic acids and a 4-channel real-time PCR module. Users can configure this system to support a variety of PCR master mixes, primer/probe combinations and control templates. An update on progress towards fielding a PCR- enabled ESP will be given along with an outline of plans for its use in coastal and oligotrophic oceanic regimes.

  19. Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

    PubMed

    Cho, Seung-Hak; Kang, Suk-Ho; Lee, Yea-Eun; Kim, Sung-Jo; Yoo, Young-Bin; Bak, Yeong-Seok; Kim, Jung-Beom

    2015-12-28

    Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

  20. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation.

    PubMed

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  1. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

    PubMed Central

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  2. Prolactin (PRL) and prolactin receptor (PRLR) genes and their role in poultry production traits.

    PubMed

    Wilkanowska, Anna; Mazurowski, Artur; Mroczkowski, Sławomir; Kokoszyński, Dariusz

    2014-01-01

    Prolactin (PRL), secreted from the anterior pituitary, plays extensive roles in osmoregulation, corpus luteum formation, mammogenesis, lactogenesis, lactopoiesis, and production of crop milk. In birds, prolactin (PRL) is generally accepted as crucial to the onset and maintenance of broodiness. All the actions of prolactin (PRL) hormone are mediated by its receptor (PRLR), which plays an important role in the PRL signal transduction cascade. It has been well established that the PRL gene is closely associated to the onset and maintenance of broody behavior, and could be a genetic marker in breeding against broodiness in chickens. Meanwhile, the prolactin receptor (PRLR) gene is regarded as a candidate genetic marker for reproductive traits. PRLR is also an important regulator gene for cell growth and differentiation. The identified polymorphism of this gene is mainly viewed in terms of egg production traits. Due to different biological activities attributed to PRL and PRLR, they can be used as major candidate genes in molecular animal breeding programs. Characterization of PRL and PRLR genes helps to elucidate their roles in birds and provides insights into the regulatory mechanisms of PRL and PRLR expression conserved in birds and mammals.

  3. Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell; Alterthum, Flavio

    1991-01-01

    A novel operon and plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase activities of Zymomonas mobilis are described. Also disclosed are methods for increasing the growth of microorganisms or eukaryotic cells and methods for reducing the accumulation of undesirable metabolic products in the growth medium of microorganisms or cells.

  4. Role of nitric oxide and flavohemoglobin homolog genes in Aspergillus nidulans sexual development and mycotoxin production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavohemoglobins are widely distributed proteins in both prokaryotic and eukaryotic organisms, conferring resistance against nitrosative stress. In the present study we investigated the role of two flavohemoglobin homologous genes, fhbA and fhbB, in morphogenesis and in the production of the mycotox...

  5. Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. ...

  6. EpsA is an essential gene in exopolysaccharide production in L actobacillus johnsonii FI9785

    PubMed Central

    Dertli, Enes; Colquhoun, Ian J.; Narbad, Arjan

    2015-01-01

    Summary L actobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)‐1 and heteropolymeric EPS‐2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS‐1 and EPS‐2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild‐type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS‐2 as the (1,4)‐linked βGlcp unit, with the acetyl group located at O‐6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression. PMID:26401596

  7. Engineering a regulatory region of jadomycin gene cluster to improve jadomycin B production in Streptomyces venezuelae.

    PubMed

    Zheng, Jian-Ting; Wang, Sheng-Lan; Yang, Ke-Qian

    2007-09-01

    Streptomyces venezuelae ISP5230 produces a group of jadomycin congeners with cytotoxic activities. To improve jadomycin fermentation process, a genetic engineering strategy was designed to replace a 3.4-kb regulatory region of jad gene cluster that contains four regulatory genes (3' end 272 bp of jadW2, jadW3, jadR2, and jadR1) and the native promoter upstream of jadJ (P(J)) with the ermEp* promoter sequence so that ermEp* drives the expression of the jadomycin biosynthetic genes from jadJ in the engineered strain. As expected, the mutant strain produced jadomycin B without ethanol treatment, and the yield increased to about twofold that of the stressed wild-type. These results indicated that manipulation of the regulation of a biosynthetic gene cluster is an effective strategy to increase product yield.

  8. [A tetracycline-inducible gene system allows for regulated production of proteins with therapeutic potential].

    PubMed

    Castillo-Ureta, Hipólito; Barrera-Saldaña, Hugo A; Martínez-Rodríguez, Herminia G

    2004-01-01

    The adoption of a bacterial system of control of genic expression with tetracycline, combined with the advances in the identification of regulatory sequences and mechanisms of expression of eukaryotic genes, has increased the versatility and effectiveness of techniques to reintroduce and to make genes function in cells and in superior organisms by being able to control the activity of the transgene in a temporary or reversible manner. This approach has also facilitated making detailed studies of different cellular processes and diseases; for example, the study of the function of oncogenes and other genes involved in the formation and progression of cancer, the generation of cellular models for recombinant protein production with therapeutic purposes, the ex-vivo genetic manipulation of extracted cells from patients and returned to them for gene therapy procedures, as well as the modulation of transgenes to revert hereditary suffering in animal models.

  9. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

    PubMed Central

    2013-01-01

    Background In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled. Results Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized. Conclusions Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment. PMID:23497240

  10. The Gene bldA, a regulator of morphological differentiation and antibiotic production in streptomyces.

    PubMed

    Hackl, Stefanie; Bechthold, Andreas

    2015-07-01

    Streptomyces species are well known for their particular features of morphological differentiation. On solid agar, a mold-like aerial mycelium is formed and spores are produced, in which the bld genes play a crucial role. In S. coelicolor, mutations in one specific bld gene called bldA led to a "naked" phenotype lacking aerial hyphae and spores. This peculiar behavior became a major interest for scientific research in the past and it was revealed that bldA is coding for a unique tRNA able to translate a UUA codon into the amino acid leucine. UUA codons are a very rare property of G + C-rich Streptomyces genomes. The impact of bldA on morphology can in parts be attributed to the regulatory effect of bldA on the translational level, because TTA-containing genes can only be translated into their corresponding protein in the presence of a fully functioning bldA gene. In addition to the visible effect of bldA expression on the phenotype of S. coelicolor, bldA mutants were also deficient in antibiotic production. This led to the assumption that the role of bldA must exceed translational control. Many TTA-containing genes are coding for transcriptional regulators which are activating or repressing the transcription of many more genes. Proteomics and transcriptomics are two powerful methods for identifying bldA target genes and it was possible to assign also post-translational regulation to bldA. This review wants to give a short overview on the importance of bldA as a regulator of morphological differentiation and antibiotic production by switching on "silent" gene clusters in Streptomyces.

  11. Consistent quantitative gene product expression: #2. Antigen intensities on bone marrow cells are invariant between individuals

    PubMed Central

    Voigt, Andrew P.; Eidenschink Brodersen, Lisa; Fritschle, Wayne; Menssen, Andrew J.; Meshinchi, Soheil; Wells, Denise A.

    2016-01-01

    Abstract Five reference populations in bone marrow specimens were identified by flow cytometry using specific combinations of reagents in order define the variation of gene product expression intensities both within and between individuals. Mature lymphocytes, uncommitted progenitor cells, promyelocytes, mature monocytes and mature neutrophils can be reproducibly identified as distinct clusters of events in heterogeneous, maturing bone marrow specimens. Support Vector Machines were used to identify the reference populations in order to reduce subjective bias in manually defining boundaries of these populations since they were not discretely separated from the remainder of the cells. Reference populations were identified in 50 randomly selected bone marrow aspirates obtained over a period spanning 3 years and 6 months from pediatric patients following chemotherapy for acute myeloid leukemia (AML). The quantitative expression of gene products (cell surface antigens) and light scattering characteristics on these stressed specimens were demonstrated to be tightly regulated both within individuals and between individuals. Within an individual most gene products (CD45, CD34, CD14, CD16, CD64, CD33) demonstrated limited variability with a standard deviation of <0.20 log units while CD13 and CD36 exhibited broader variation >0.25 log units. Surprisingly, with the exception of CD33, the variation of the mean intensities of each antigen between individuals was even less than the variation within an individual. These data confirm that the amounts of gene products expressed on normal developing cells are highly regulated but differ in intensities between different lineages and during the maturational pathway of those lineages. The amounts of gene products expressed at specific stages of development of each lineage are a biologic constant with minimal variation within or between individuals. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of

  12. Analysis of ldh genes in Lactobacillus casei BL23: role on lactic acid production.

    PubMed

    Rico, Juan; Yebra, María Jesús; Pérez-Martínez, Gaspar; Deutscher, Josef; Monedero, Vicente

    2008-06-01

    Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A Deltaldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a Deltaldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid.

  13. Functional Reconstitution of a Fungal Natural Product Gene Cluster by Advanced Genome Editing.

    PubMed

    Weber, Jakob; Valiante, Vito; Nødvig, Christina S; Mattern, Derek J; Slotkowski, Rebecca A; Mortensen, Uffe H; Brakhage, Axel A

    2017-01-20

    Filamentous fungi produce varieties of natural products even in a strain dependent manner. However, the genetic basis of chemical speciation between strains is still widely unknown. One example is trypacidin, a natural product of the opportunistic human pathogen Aspergillus fumigatus, which is not produced among different isolates. Combining computational analysis with targeted gene editing, we could link a single nucleotide insertion in the polyketide synthase of the trypacidin biosynthetic pathway and reconstitute its production in a nonproducing strain. Thus, we present a CRISPR/Cas9-based tool for advanced molecular genetic studies in filamentous fungi, exploiting selectable markers separated from the edited locus.

  14. The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI.

    PubMed

    Scholthof, H B; Gowda, S; Wu, F C; Shepherd, R J

    1992-05-01

    Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes.

  15. Modern plant metabolomics: Advanced natural product gene discoveries, improved technologies, and future prospects

    SciTech Connect

    Sumner, Lloyd W.; Lei, Zhentian; Nikolau, Basil J.; Saito, Kazuki

    2014-10-24

    Plant metabolomics has matured and modern plant metabolomics has accelerated gene discoveries and the elucidation of a variety of plant natural product biosynthetic pathways. This study highlights specific examples of the discovery and characterization of novel genes and enzymes associated with the biosynthesis of natural products such as flavonoids, glucosinolates, terpenoids, and alkaloids. Additional examples of the integration of metabolomics with genome-based functional characterizations of plant natural products that are important to modern pharmaceutical technology are also reviewed. This article also provides a substantial review of recent technical advances in mass spectrometry imaging, nuclear magnetic resonance imaging, integrated LC-MS-SPE-NMR for metabolite identifications, and x-ray crystallography of microgram quantities for structural determinations. The review closes with a discussion on the future prospects of metabolomics related to crop species and herbal medicine.

  16. Modern plant metabolomics: Advanced natural product gene discoveries, improved technologies, and future prospects

    DOE PAGES

    Sumner, Lloyd W.; Lei, Zhentian; Nikolau, Basil J.; ...

    2014-10-24

    Plant metabolomics has matured and modern plant metabolomics has accelerated gene discoveries and the elucidation of a variety of plant natural product biosynthetic pathways. This study highlights specific examples of the discovery and characterization of novel genes and enzymes associated with the biosynthesis of natural products such as flavonoids, glucosinolates, terpenoids, and alkaloids. Additional examples of the integration of metabolomics with genome-based functional characterizations of plant natural products that are important to modern pharmaceutical technology are also reviewed. This article also provides a substantial review of recent technical advances in mass spectrometry imaging, nuclear magnetic resonance imaging, integrated LC-MS-SPE-NMR formore » metabolite identifications, and x-ray crystallography of microgram quantities for structural determinations. The review closes with a discussion on the future prospects of metabolomics related to crop species and herbal medicine.« less

  17. Correlation of gene expression and protein production rate - a system wide study

    PubMed Central

    2011-01-01

    Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR). PMID:22185473

  18. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes

    PubMed Central

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-01-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l−1) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l−1 h−1. Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. PMID:25488800

  19. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

    PubMed

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-03-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1)  h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol.

  20. 1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

    PubMed

    Przystałowska, Hanna; Zeyland, Joanna; Szymanowska-Powałowska, Daria; Szalata, Marlena; Słomski, Ryszard; Lipiński, Daniel

    2015-02-01

    1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol₁,₃-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming

  1. Decarboxylase gene expression and cadaverine and putrescine production by Serratia proteamaculans in vitro and in beef.

    PubMed

    De Filippis, Francesca; Pennacchia, Carmela; Di Pasqua, Rosangela; Fiore, Alberto; Fogliano, Vincenzo; Villani, Francesco; Ercolini, Danilo

    2013-08-01

    Studies of the molecular basis of microbial metabolic activities that are important for the changes in food quality are valuable in order to help in understanding the behavior of spoiling bacteria in food. The growth of a psychrotrophic Serratia proteamaculans strain was monitored in vitro and in artificially inoculated raw beef. Two growth temperatures (25°C and 4°C) were tested in vitro, while growth at 15°C and 4°C was monitored in beef. During growth, the expression of inducible lysine and ornithine-decarboxylase genes was evaluated by quantitative reverse transcription-PCR (qRT-PCR), while the presence of cadaverine and putrescine was quantified by LC-ESI-MS/MS. The expression of the decarboxylase genes, and the consequent production of cadaverine and putrescine were shown to be influenced by the temperature, as well as by the complexity of the growth medium. Generally, the maximum gene expression and amine production took place during the exponential and early stationary phase, respectively. In addition, lower temperatures caused slower growth and gene downregulation. Higher amounts of cadaverine compared to putrescine were found during growth in beef with the highest concentrations corresponding to microbial loads of ca. 9CFU/g. The differences found in gene expression evaluated in vitro and in beef suggested that such activities are more reliably investigated in situ in specific food matrices.

  2. Major histocompatibility complex gene product expression on pancreatic beta cells in acutely diabetic BB rats.

    PubMed Central

    Issa-Chergui, B.; Yale, J. F.; Vigeant, C.; Seemayer, T. A.

    1988-01-01

    Type I diabetes mellitus was induced in young, diabetes-prone BB rats by the passive transfer of concanavalin A-activated T lymphocytes from the spleens of acutely diabetic BB rats. The pancreas of the recipients was examined 1-2 days after the onset of glycosuria by immunocytochemistry by means of monoclonal antibodies for determining whether 1) Class I and/or II major histocompatibility gene complex (MHC) products were expressed on beta cells and 2) the mononuclear cell infiltrates were represented by T cells. Marked expression of Class I MHC gene products was evident on beta cells. In contrast, Class II MHC gene products were not identified on normal-appearing beta cells. Dendritic cells dispersed throughout the acinar and interstitial pancreas were markedly increased in number. The mononuclear cell infiltrate contained few cells (1-15%) recognized by a pan-T cell marker. Although it is possible that this passive transfer model might differ considerably from the spontaneously occurring diabetic state in the rat, this study suggests that 1) Class I, rather than Class II, MHC gene expression may be pivotal to beta-cell injury in diabetic rats, and 2) non-T cells may constitute an effector cell population central to beta-cell necrosis in Type I diabetes mellitus. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:3276208

  3. Identification of the phoM gene product and its regulation in Escherichia coli K-12.

    PubMed Central

    Ludtke, D; Bernstein, J; Hamilton, C; Torriani, A

    1984-01-01

    Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain. Images PMID:6330029

  4. Association of methionine synthase gene polymorphisms with wool production and quality traits in Chinese Merino population.

    PubMed

    Rong, E G; Yang, H; Zhang, Z W; Wang, Z P; Yan, X H; Li, H; Wang, N

    2015-10-01

    Methionine synthase (MTR) plays a crucial role in maintaining homeostasis of intracellular methionine, folate, and homocysteine, and its activity correlates with DNA methylation in many mammalian tissues. Our previous genomewide association study identified that 1 SNP located in the gene was associated with several wool production and quality traits in Chinese Merino. To confirm the potential involvement of the gene in sheep wool production and quality traits, we performed sheep tissue expression profiling, SNP detection, and association analysis with sheep wool production and quality traits. The semiquantitative reverse transcription PCR analysis showed that the gene was differentially expressed in skin from Merino and Kazak sheep. The sequencing analysis identified a total of 13 SNP in the gene from Chinese Merino sheep. Comparison of the allele frequencies revealed that these 13 identified SNP were significantly different among the 6 tested Chinese Merino strains ( < 0.001). Linkage disequilibrium analysis showed that SNP 3 to 11 were strongly linked in a single haplotype block in the tested population. Association analysis showed that SNP 2 to 11 were significantly associated with the average wool fiber diameter and the fineness SD and that SNP 4 to 11 were significantly associated with the CV of fiber diameter trait ( < 0.05). Single nucleotide polymorphism 2 and SNP 5 to 12 were weakly associated with wool crimp. Similarly, the haplotypes derived from these 13 identified SNP were also significantly associated with the average wool fiber diameter, fineness SD, and the CV of fiber diameter ( < 0.05). Our results suggest that is a candidate gene for sheep wool production and quality traits, and the identified SNP might be used in sheep breeding.

  5. Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins.

    PubMed

    Chen, Qiang; Lai, Huafang; Hurtado, Jonathan; Stahnke, Jake; Leuzinger, Kahlin; Dent, Matthew

    2013-06-01

    Current human biologics are most commonly produced by mammalian cell culture-based fermentation technologies. However, its limited scalability and high cost prevent this platform from meeting the ever increasing global demand. Plants offer a novel alternative system for the production of pharmaceutical proteins that is more scalable, cost-effective, and safer than current expression paradigms. The recent development of deconstructed virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins, and in turn, provided a preferred plant based production platform. One of the remaining challenges for the commercial application of this platform was the lack of a scalable technology to deliver the transgene into plant cells. Therefore, this review focuses on the development of an effective and scalable technology for gene delivery in plants. Direct and indirect gene delivery strategies for plant cells are first presented, and the two major gene delivery technologies based on agroinfiltration are subsequently discussed. Furthermore, the advantages of syringe and vacuum infiltration as gene delivery methodologies are extensively discussed, in context of their applications and scalability for commercial production of human pharmaceutical proteins in plants. The important steps and critical parameters for the successful implementation of these strategies are also detailed in the review. Overall, agroinfiltration based on syringe and vacuum infiltration provides an efficient, robust and scalable gene-delivery technology for the transient expression of recombinant proteins in plants. The development of this technology will greatly facilitate the realization of plant transient expression systems as a premier platform for commercial production of pharmaceutical proteins.

  6. [Adeno-associated viral vectors: methods for production and purification for gene therapy applications].

    PubMed

    Mena-Enriquez, Mayra; Flores-Contreras, Lucia; Armendáriz-Borunda, Juan

    2012-01-01

    Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials.

  7. Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

    SciTech Connect

    Kenney, S.; Kamine, J.; Markovitz, D.; Fenrick, R.; Pagano, J.

    1988-03-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

  8. The FHIT gene product: tumor suppressor and genome ‘caretaker’

    PubMed Central

    Waters, Catherine E.; Saldivar, Joshua C.; Hosseini, Seyed Ali; Huebner, Kay

    2014-01-01

    The FHIT gene at FRA3B is one of the earliest and most frequently altered genes in the majority of human cancers. It was recently discovered that the FHIT gene is not the most fragile locus in epithelial cells, the cell of origin for most Fhit negative cancers, eroding support for past claims that deletions at this locus are simply passenger events that are carried along in expanding cancer clones, due to extreme vulnerability to DNA damage rather than to loss of FHIT function. Indeed, recent reports have reconfirmed FHIT as a tumor suppressor gene with roles in apoptosis and prevention of the epithelial-mesenchymal transition. Other recent works have identified a novel role for the FHIT gene product, Fhit, as a genome ‘caretaker.’ Loss of this caretaker function leads to nucleotide imbalance, spontaneous replication stress, and DNA breaks. Because Fhit loss-induced DNA damage is “checkpoint blind,” cells accumulate further DNA damage during subsequent cell cycles, accruing global genome instability that could facilitate oncogenic mutation acquisition and expedite clonal expansion. Loss of Fhit activity therefore induces a mutator phenotype. Evidence for FHIT as a mutator gene is discussed in light of these recent investigations of Fhit loss and subsequent genome instability. PMID:25283145

  9. Glutamic acid promotes monacolin K production and monacolin K biosynthetic gene cluster expression in Monascus.

    PubMed

    Zhang, Chan; Liang, Jian; Yang, Le; Chai, Shiyuan; Zhang, Chenxi; Sun, Baoguo; Wang, Chengtao

    2017-12-01

    This study investigated the effects of glutamic acid on production of monacolin K and expression of the monacolin K biosynthetic gene cluster. When Monascus M1 was grown in glutamic medium instead of in the original medium, monacolin K production increased from 48.4 to 215.4 mg l(-1), monacolin K production increased by 3.5 times. Glutamic acid enhanced monacolin K production by upregulating the expression of mokB-mokI; on day 8, the expression level of mokA tended to decrease by Reverse Transcription-polymerase Chain Reaction. Our findings demonstrated that mokA was not a key gene responsible for the quantity of monacolin K production in the presence of glutamic acid. Observation of Monascus mycelium morphology using Scanning Electron Microscope showed glutamic acid significantly increased the content of Monascus mycelium, altered the permeability of Monascus mycelium, enhanced secretion of monacolin K from the cell, and reduced the monacolin K content in Monascus mycelium, thereby enhancing monacolin K production.

  10. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts.

    PubMed

    Malpass, Gloria E; Arimilli, Subhashini; Prasad, G L; Howlett, Allyn C

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1h or 5h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5h), which was increased by nicotine but suppressed by other components of STE. Within 2h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research.

  11. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    PubMed

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  12. The SKP1-like gene family of Arabidopsis exhibits a high degree of differential gene expression and gene product interaction during development.

    PubMed

    Dezfulian, Mohammad H; Soulliere, Danielle M; Dhaliwal, Rajdeep K; Sareen, Madhulika; Crosby, William L

    2012-01-01

    The Arabidopsis thaliana genome encodes several families of polypeptides that are known or predicted to participate in the formation of the SCF-class of E3-ubiquitin ligase complexes. One such gene family encodes the Skp1-like class of polypeptide subunits, where 21 genes have been identified and are known to be expressed in Arabidopsis. Phylogenetic analysis based on deduced polypeptide sequence organizes the family of ASK proteins into 7 clades. The complexity of the ASK gene family, together with the close structural similarity among its members raises the prospect of significant functional redundancy among select paralogs. We have assessed the potential for functional redundancy within the ASK gene family by analyzing an expanded set of criteria that define redundancy with higher resolution. The criteria used include quantitative expression of locus-specific transcripts using qRT-PCR, assessment of the sub-cellular localization of individual ASK:YFP auto-fluorescent fusion proteins expressed in vivo as well as the in planta assessment of individual ASK-F-Box protein interactions using bimolecular fluorescent complementation techniques in combination with confocal imagery in live cells. The results indicate significant functional divergence of steady state transcript abundance and protein-protein interaction specificity involving ASK proteins in a pattern that is poorly predicted by sequence-based phylogeny. The information emerging from this and related studies will prove important for defining the functional intersection of expression, localization and gene product interaction that better predicts the formation of discrete SCF complexes, as a prelude to investigating their molecular mode of action.

  13. PepPSy: a web server to prioritize gene products in experimental and biocuration workflows

    PubMed Central

    Sallou, Olivier; Duek, Paula D.; Darde, Thomas A.; Collin, Olivier; Lane, Lydie; Chalmel, Frédéric

    2016-01-01

    Among the 20 000 human gene products predicted from genome annotation, about 3000 still lack validation at protein level. We developed PepPSy, a user-friendly gene expression-based prioritization system, to help investigators to determine in which human tissues they should look for an unseen protein. PepPSy can also be used by biocurators to revisit the annotation of specific categories of proteins based on the ‘omics’ data housed by the system. In this study, it was used to prioritize 21 dubious protein-coding genes among the 616 annotated in neXtProt for reannotation. PepPSy is freely available at http://peppsy.genouest.org. Database URL: http://peppsy.genouest.org. PMID:27173522

  14. Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing.

    PubMed

    Wu, Hong; Watanabe, Tomoko; Araki, Yoshio; Kitagaki, Hiroshi; Akao, Takeshi; Takagi, Hiroshi; Shimoi, Hitoshi

    2009-06-01

    Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.

  15. Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene.

    PubMed

    Cha, Mijeong; Shim, Sang Hee; Kim, Sung Hong; Kim, Ok Tae; Lee, Se-Weon; Kwon, Suk-Yoon; Baek, Kwang-Hyun

    2012-10-01

    Paclitaxel is produced by various species of yew trees and has been extensively used to treat tumors. In our research, a taxadiene synthase (TS) gene from Taxus brevifolia was used to transform the roots of cultured ginseng (Panax ginseng C.A. Meyer) to produce taxadiene, the unique skeletal precursor to taxol. The TS gene was successfully introduced into the ginseng genome, and the de novo formation of taxadiene was identified by mass spectroscopy profiling. Without any change in phenotypes or growth difference in a TS-transgenic ginseng line, the transgenic TSS3-2 line accumulated 9.1 μg taxadiene per gram of dry weight. In response to the treatment of methyl jasmonate for 3 or 6 days, the accumulation was 14.6 and 15.9 μg per g of dry weight, respectively. This is the first report of the production of taxadiene by engineering ginseng roots with a taxadiene synthase gene.

  16. Localization of the mei-1 gene product of Caenorhaditis elegans, a meiotic-specific spindle component

    PubMed Central

    1994-01-01

    Genetic evidence suggests that the product of the mei-1 gene of Caenorhabditis elegans is specifically required for meiosis in the female germline. Loss-of-function mei-1 mutations block meiotic spindle formation while a gain-of-function allele instead results in spindle defects during the early mitotic cleavages. In this report, we use immunocytochemistry to examine the localization of the mei-1 product in wild-type and mutant embryos. During metaphase of meiosis I in wild- type embryos, mei-1 protein was found throughout the spindle but was more concentrated toward the poles. At telophase I, mei-1 product colocalized with the chromatin at the spindle poles. The pattern was repeated during meiosis II but no mei-1 product was visible during the subsequent mitotic cleavages. The mei-1 gain-of-function allele resulted in ectopic mei-1 staining in the centers of the microtubule- organizing centers during interphase and in the spindles during the early cleavages. This aberrant localization is probably responsible for the poorly formed and misoriented cleavage spindles characteristic of the mutation. We also examined the localization of mei-1(+) product in the presence of mutations of genes that genetically interact with mei-1 alleles. mei-2 is apparently required to localize mei-1 product to the spindle during meiosis while mel-26 acts as a postmeiotic inhibitor. We conclude that mei-1 encodes a novel spindle component, one that is specialized for the acentriolar meiotic spindles unique to female meiosis. The genes mei-2 and mel-26 are part of a regulatory network that confines mei-1 activity to meiosis. PMID:8027178

  17. EMEA and Gene Therapy Medicinal Products Development in the European Union

    PubMed Central

    2003-01-01

    The evaluation of quality, safety, and efficacy of medicinal products by the European Medicines Evaluation Agency (EMEA) via the centralized procedure is the only available regulatory procedure for obtaining marketing authorization for gene therapy (GT) medicinal products in the European Union. The responsibility for the authorization of clinical trials remains with the national competent authorities (NCA) acting in a harmonized framework from the scientific viewpoint. With the entry into force of a new directive on good clinical practice implementation in clinical trials as of 1 May 2004, procedural aspects will also be harmonized at EU level. Scientifically sound development of medicinal products is the key for the successful registration of dossiers and for contributing to the promotion and protection of public health. The objective of this paper is to introduce the EMEA regulatory processes and scientific activities relevant to GT medicinal products. PMID:12686717

  18. Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene.

    PubMed

    Kahraman, Huseyin; Aytan, Emel; Kurt, Ash Giray

    2011-09-01

    The production of antileukemic enzyme methionine γ-lyase (MGL) in distinctly related bacteria, Citrobacter freundii and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. This study concerns the potential of Citrobacter freundii expressing the Vitreoscilla hemoglobin gene (vgb) for the methionine γ- liyase production. Methionine γ- liyase production by Citrobacter freundii and its vgb(-) and vgb(+) bearing recombinant strain was studied in shake-flasks under 200 rpm agitation, culture medium and 30 °C in a time-course manner. The vgb(+) and especially the carbon type had a dramatic effect on methionine γ- liyase production. The vgb(+) strain of C. freundii had about 2-fold and 3.1-fold higher levels of MGL than the host and vgb(-) strain, respectively.

  19. A repressor-response regulator gene pair controlling jadomycin B production in Streptomyces venezuelae ISP5230.

    PubMed

    Yang, K; Han, L; He, J; Wang, L; Vining, L C

    2001-11-28

    A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.

  20. Regulation of Aspergillus genes encoding plant cell wall polysaccharide-degrading enzymes; relevance for industrial production.

    PubMed

    de Vries, R P

    2003-03-01

    The genus Aspergillus is widely used for the production of plant cell wall polysaccharide-degrading enzymes. The range of enzymes purified from these fungi covers nearly every function required for the complete degradation of cellulose, xyloglucan, xylan, galacto(gluco)mannan and pectin. This paper describes the Aspergillus enzymes involved in the degradation of these polysaccharides and discusses the regulatory systems involved in the expression of the genes encoding these proteins. The latter is of major importance in the large-scale production of these enzymes for industrial applications.

  1. Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus▿ †

    PubMed Central

    Lan, Lefu; Cheng, Alice; Dunman, Paul M.; Missiakas, Dominique; He, Chuan

    2010-01-01

    The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of ∼400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival. PMID:20400547

  2. TITER AND PRODUCT AFFECTS THE DISTRIBUTION OF GENE EXPRESSION AFTER INTRAPUTAMINAL CONVECTION-ENHANCED DELIVERY

    PubMed Central

    Emborg, Marina E.; Hurley, Samuel A.; Joers, Valerie; Tromp, Do P.M.; Swanson, Christine R.; Ohshima-Hosoyama, Sachiko; Bondarenko, Viktorya; Cummisford, Kyle; Sonnemans, Marc; Hermening, Stephan; Blits, Bas; Alexander, Andrew L.

    2014-01-01

    Background Efficacy and safety of intracerebral gene therapy for brain disorders, like Parkinson’s disease, depends on appropriate distribution of gene expression. Objectives To assess if the distribution of gene expression is affected by vector titer and protein type. Methods Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received in the right and left ventral postcommisural putamen 30μl inoculation of a high or low titer suspension of AAV5 encoding glial derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Inoculations were performed using convection enhanced delivery and intraoperative MRI (IMRI). Results IMRI confirmed targeting and infusion cloud irradiating from the catheter tip into surrounding area. Postmortem analysis six weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection side that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the Substantia Nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many SNpc and SNpr neurons. Conclusions After controlling for target and infusate volume, intracerebral distribution of gene product is affected by vector titer and product biology. PMID:24943657

  3. Effects of Copper on Hemocyte Apoptosis, ROS Production, and Gene Expression in White Shrimp Litopenaeus vannamei.

    PubMed

    Guo, Hui; Li, Kexu; Wang, Wei; Wang, Chenggui; Shen, Yuchun

    2017-02-25

    Copper, a common chemical contaminant in aquatic environment, is known to be toxic to aquatic life at high concentrations. In the present study, we evaluated the apoptotic cell ratio and ROS production in hemocytes of the white shrimp Litopenaeus vannamei exposed to 1 or 5 mg L(-1) Cu for 0, 3, 6, 12, 24, and 48 h. The expression changes of antioxidant biomarker genes, i.e., copper-zinc superoxide dismutase (Cu-Zn SOD) and catalase (CAT), apoptosis-related genes, i.e., caspase-3 and inhibitor of apoptosis protein (IAP), and a specific biomarker gene of heavy metal pollution, i.e., metallothionein (MT), were also determined in hemocytes. Significant increases in ROS production were observed in both treatment groups at each time points. The apoptotic cell ratios were significantly increased at 6-48 h among shrimp exposed to 1 mg L(-1) Cu and at each time points in 5 mg L(-1) Cu group. These results indicated that Cu would induce oxidative stress and apoptosis in the hemocyte of L. vannamei. Quantitative real-time PCR analysis revealed that the relative expression levels of Cu-Zn SOD, CAT, caspase-3, IAP, and MT were upregulated in a dose-dependent and time-dependent manner, suggesting the involvement of these genes in stress response against Cu exposure.

  4. Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control.

    PubMed

    Perkin, Lindsey C; Adrianos, Sherry L; Oppert, Brenda

    2016-09-19

    Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs.

  5. Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control

    PubMed Central

    Perkin, Lindsey C.; Adrianos, Sherry L.; Oppert, Brenda

    2016-01-01

    Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs. PMID:27657138

  6. Induction of mitotic gene conversion by browning reaction products and its modulation by naturally occurring agents.

    PubMed

    Rosin, M P; Stich, H F; Powrie, W D; Wu, C H

    1982-05-01

    Mitotic gene conversion in the D7 strain of Saccharomyces cerevisiae was significantly enhanced by exposure to non-enzymatic browning reaction products. These products were formed during the heating of sugar (caramelization reaction) or sugar-amino acid mixtures (Maillard reaction) at temperatures normally used during the cooking of food. Several modulating factors of this convertogenic activity were identified. These factors included two main groups: (1) trace metals which are widely distributed in the environment; and (2) several cellular enzymatic systems. The convertogenic activities of a heated glucose-lysine mixture and a commercial caramel powder were completely suppresses when yeast were concurrently exposed to these products and to either FeIII or CuII. Equimolar concentrations of MnII or sodium selenite had no effect on the convertogenic activity of the products of either model system. Horse-radish peroxidase, beef liver catalase and rat liver S9 preparations each decreased the frequency of gene conversion induced by the caramel powder and the heated glucose-lysine products. This modulating activity of the enzymes was lost if they were heat-inactivated. These studies indicate the presence of a variety of protective mechanisms which can modify genotoxic components in complex food mixtures.

  7. Solid-phase peptide head-to-side chain cyclodimerization: discovery of C(2)-symmetric cyclic lactam hybrid α-melanocyte-stimulating hormone (MSH)/agouti-signaling protein (ASIP) analogues with potent activities at the human melanocortin receptors.

    PubMed

    Mayorov, Alexander V; Cai, Minying; Palmer, Erin S; Liu, Zhihua; Cain, James P; Vagner, Josef; Trivedi, Dev; Hruby, Victor J

    2010-10-01

    A novel hybrid melanocortin pharmacophore was designed based on the pharmacophores of the agouti-signaling protein (ASIP), an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. The designed hybrid ASIP/MSH pharmacophore was explored in monomeric cyclic, and cyclodimeric templates. The monomeric cyclic disulfide series yielded peptides with hMC3R-selective non-competitive binding affinities. The direct on-resin peptide lactam cyclodimerization yielded nanomolar range (25-120 nM) hMC1R-selective full and partial agonists in the cyclodimeric lactam series which demonstrates an improvement over the previous attempts at hybridization of MSH and agouti protein sequences. The secondary structure-oriented pharmacophore hybridization strategy will prove useful in development of unique allosteric and orthosteric melanocortin receptor modulators. This report also illustrates the utility of peptide cyclodimerization for the development of novel GPCR peptide ligands.

  8. Description of a new species of Ixodes Latreille, 1795 (Acari: Ixodidae) and redescription of I. lasallei Méndez & Ortiz, 1958, parasites of agoutis and pacas (Rodentia: Dasyproctidae, Cuniculidae) in Central and South America.

    PubMed

    Apanaskevich, Dmitry A; Bermúdez, Sergio E

    2017-05-01

    Ixodes bocatorensis n. sp. (Acari: Ixodidae), is described based on adults ex agoutis (Rodentia: Dasyproctidae), pacas (Rodentia: Cuniculidae) and "tapir and sloth" (Perissodactyla: Tapiridae and Pilosa) from Colombia, Panama and Venezuela. Adults of I. bocatorensis n. sp. are similar to those of I. lasallei Méndez & Ortiz, 1958 but can be distinguished by the scutum dimensions, punctation pattern, gnathosoma and palpi measurements and their ratios, basis capituli anterior angle and shape of the spur of palpal segment I ventrally. For comparative purposes the female of I. lasallei is redescribed and the true male of this species is described for the first time. Studied adults of I. lasallei were found on agoutis, pacas and ocelot (Carnivora: Felidae) in Colombia, Peru and Venezuela.

  9. Identification of the Three Genes Involved in Controlling Production of a Phytotoxin Tropolone in Burkholderia plantarii

    PubMed Central

    Miwa, Shunpei; Kihira, Eri; Yoshioka, Akinori; Nakasone, Kaoru; Okamoto, Sho; Hatano, Masaki; Igarashi, Masayuki; Eguchi, Yoko; Kato, Akinori; Ichikawa, Natsuko; Sekine, Mitsuo; Fujita, Nobuyuki; Kanesaki, Yu; Yoshikawa, Hirofumi

    2016-01-01

    ABSTRACT Tropolone, a phytotoxin produced by Burkholderia plantarii, causes rice seedling blight. To identify genes involved in tropolone synthesis, we systematically constructed mutations in the genes encoding 55 histidine kinases and 72 response regulators. From the resulting defective strains, we isolated three mutants, KE1, KE2, and KE3, in which tropolone production was repressed. The deleted genes of these mutants were named troR1, troK, and troR2, respectively. The mutant strains did not cause rice seedling blight, and complementation experiments indicated that TroR1, TroK, and TroR2 were involved in the synthesis of tropolone in B. plantarii. However, tropolone synthesis was repressed in the TroR1 D52A, TroK H253A, and TroR2 D46A site-directed mutants. These results suggest that the putative sensor kinase (TroK) and two response regulators (TroR1 and TroR2) control the production of tropolone in B. plantarii. IMPORTANCE A two-component system is normally composed of a sensor histidine kinase (HK) and a cognate response regulator (RR) pair. In this study, HK (TroK) and two RRs (TroR1 and TroR2) were found to be involved in controlling tropolone production in B. plantarii. These three genes may be part of a bacterial signal transduction network. Such networks are thought to exist in other bacteria to regulate phytotoxin production, as well as environmental adaptation and signal transduction. PMID:27002128

  10. Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes.

    PubMed

    Wei, Yongjun; Gossing, Michael; Bergenholm, David; Siewers, Verena; Nielsen, Jens

    2017-12-01

    Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0-C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0), but CB supply is limited. Therefore, CB-like lipids (CBL, which are composed of POP, POS and SOS) are in great demand. Saccharomyces cerevisiae produces TAGs as storage lipids, which are also mainly composed of C16 and C18 fatty acids. However, POP, POS and SOS are not among the major TAG forms in yeast. TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast strains harboring cocoa genes increased 190, 230 and 196% over the control strain, respectively; especially, the potential SOS content of the three yeast strains increased 254, 476 and 354% over the control strain. Moreover, one of the three yeast strains had a 2.25-fold increased TAG content and 6.7-fold higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis.

  11. Genetic resources for methane production from biomass described with the Gene Ontology

    PubMed Central

    Purwantini, Endang; Torto-Alalibo, Trudy; Lomax, Jane; Setubal, João C.; Tyler, Brett M.; Mukhopadhyay, Biswarup

    2014-01-01

    Methane (CH4) is a valuable fuel, constituting 70–95% of natural gas, and a potent greenhouse gas. Release of CH4 into the atmosphere contributes to climate change. Biological CH4 production or methanogenesis is mostly performed by methanogens, a group of strictly anaerobic archaea. The direct substrates for methanogenesis are H2 plus CO2, acetate, formate, methylamines, methanol, methyl sulfides, and ethanol or a secondary alcohol plus CO2. In numerous anaerobic niches in nature, methanogenesis facilitates mineralization of complex biopolymers such as carbohydrates, lipids and proteins generated by primary producers. Thus, methanogens are critical players in the global carbon cycle. The same process is used in anaerobic treatment of municipal, industrial and agricultural wastes, reducing the biological pollutants in the wastes and generating methane. It also holds potential for commercial production of natural gas from renewable resources. This process operates in digestive systems of many animals, including cattle, and humans. In contrast, in deep-sea hydrothermal vents methanogenesis is a primary production process, allowing chemosynthesis of biomaterials from H2 plus CO2. In this report we present Gene Ontology (GO) terms that can be used to describe processes, functions and cellular components involved in methanogenic biodegradation and biosynthesis of specialized coenzymes that methanogens use. Some of these GO terms were previously available and the rest were generated in our Microbial Energy Gene Ontology (MENGO) project. A recently discovered non-canonical CH4 production process is also described. We have performed manual GO annotation of selected methanogenesis genes, based on experimental evidence, providing “gold standards” for machine annotation and automated discovery of methanogenesis genes or systems in diverse genomes. Most of the GO-related information presented in this report is available at the MENGO website (http

  12. CAPA-Gene Products in the Haematophagous Sandfly Phlebotomus papatasi (Scopoli) - Vector for Leishmaniasis Disease

    DTIC Science & Technology

    2012-01-01

    Please cite this article in press as: Predel R, et al. CAPA-gene products in the haematophagous sandfly Phlebotomus papatasi (Scopoli) – vector for...the haematophagous sandfly Phlebotomus papatasi (Scopoli) – vector for leishmaniasis disease 1 2 Reinhard Predela,e,∗, Susanne Neuperta,e...neuropeptides24 Periviscerokinins25 Corazonin26 Phlebotomus papatasi27 Psychodidae28 Differential processing29 a b s t r a c t Sandflies

  13. Characterization of the Expression of Basigin Gene Products Within the Pineal Gland of Mice.

    PubMed

    Tokar, Derek; van Ekeris, Leslie; Linser, Paul J; Ochrietor, Judith D

    2016-11-04

    The expression of Basigin gene products and monocarboxylate transporter-1 (MCT1) has been investigated within the mammalian neural retina and suggests a role for these proteins in cellular metabolism within that tissue. The purpose of the present study was to investigate the expression of these same proteins in the pineal gland of the mouse brain. Mouse pineal gland and neural retina RNA and protein were subjected to quantitative reverse transcription-polymerase chain reaction and immunoblotting analyses. In addition, paraffin-embedded sections of each tissue were analyzed for expression of Basigin gene products and MCT1 via immunohistochemistry. The results indicate that MCT1 and Basigin variant-2, but not Basigin variant-1, are expressed within the mouse pineal gland. The expression of Basigin variant-2 and MCT1 was localized to the capsule surrounding the gland. The position and relative amounts of the gene products suggest that they play a much less prominent role within the pineal gland than in the neural retina.

  14. Diversity of Streptococcus thermophilus in bacteriocin production; inhibitory spectrum and occurrence of thermophilin genes.

    PubMed

    Rossi, Franca; Marzotto, Marta; Cremonese, Silvia; Rizzotti, Lucia; Torriani, Sandra

    2013-08-01

    The bacteriocin-producing Streptococcus thermophilus strains that can dominate in natural dairy ecosystems, may also enhance safety in products obtained from natural cultures. In this study, we sought to identify bacteriocin production and bacteriocin genes in 75 strains of dairy and plant origin. The strains were tested for antimicrobial activity against pathogens or pathogen models, spoiling bacteria, and lactic acid bacteria associated with dairy products. All strains moderately inhibited Staphylococcus aureus P310, none inhibited Listeria innocua LMG 11387(T) or Clostridium tyrobutyricum LMG 1285(T). In addition, 14 were active against one or more indicators in addition to S. aureus P310. Inhibition of other starter bacteria was more common than the inhibition of unwanted microorganisms. The involvement of a proteinaceous compound was ascertained in all cases. Results suggested that the selection of bacteriocinogenic S. thermophilus strains for use in biopreservation must take into account the effects exerted on other lactic acid bacteria. PCR detection of thermophilin genes proved unreliable in predicting antimicrobial activity. For S. thermophilus PRI36 and PRI45, with relevant inhibitory features, the identity of the bacteriocin genes present in the thermophilin 9 cluster was defined, thus revealing novel variants for this genome region.

  15. Identification of the Pr1 Gene Product Completes the Anthocyanin Biosynthesis Pathway of Maize

    PubMed Central

    Sharma, Mandeep; Cortes-Cruz, Moises; Ahern, Kevin R.; McMullen, Michael; Brutnell, Thomas P.; Chopra, Surinder

    2011-01-01

    In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3′H encoding gene (Zmf3′h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3′H1 promoter–gene construct established that the encoded protein product was sufficient to perform a 3′-hydroxylation reaction. The Zmf3′h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5′-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize. PMID:21385724

  16. Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus Isolates in Fishery Products in Iran

    PubMed Central

    Arfatahery, Noushin; Davoodabadi, Abolfazl; Abedimohtasab, Taranehpeimaneh

    2016-01-01

    Staphylococcus aureus is one of the most common causes of seafood-borne diseases worldwide, which are attributable to the contamination of food by preformed enterotoxins. In this study, a total of 206 (34.3%) Staphylococcus aureus strains were obtained from 600 fish and shrimp samples and were tested for their antimicrobial susceptibility. We assessed the prevalence of the genes responsible for the staphylococcal enterotoxins (SEA, SEB) and toxic shock syndrome toxin 1 (TSST-1) genes. The results indicated that 34% of aqua food samples were contaminated with S. aureus, and 23.8% of these isolates were mec-A-positive. Sixty-four percent of the strains isolated from contaminated seafood was enterotoxigenic S. aureus, and 28.2% of SEs were MRSA-positive. The most prevalent genotype was characterized by the presence of the sea gene (45.2%), followed by the seb gene (18.5%), and the tst gene encoding TSST-1 was found in eight strains (3.9%). Of the 206 S. aureus isolates, 189 strains (84.9%) were resistant to at least one antibiotic. Given the frequent outbreaks of enterotoxigenic MRSA, it is necessary to make revisions to mandatory programmes to facilitate improved hygiene practices during fishing, aquaculture, processing, and sales to prevent the contamination of fishery products in Iran. PMID:27694813

  17. Functional cooperation of the dnaE and dnaN gene products in Escherichia coli.

    PubMed Central

    Kuwabara, N; Uchida, H

    1981-01-01

    A system was designed to isolate second-site intergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the alpha subunit of DNA polymerase III [McHenry, C. S. & Crow, W. (1979) J. Biol. Chem. 254, 1748-1753]. One such suppressor, named sueA77, was finely mapped and found to be located at 82 min on the E. coli chromosome, between dnaA and recF, and within the dnaN gene [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553]. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme [Burgers, P. M. J., Kornberg, A. & Sakakibara, Y. (1981) Proc. Natl. Acad. Sci. USA 78, 5391-5395]. The sueA77 mutation was trans-dominant over its wild-type allele, and it suppressed different thermosensitive mutations of dnaE with different maximal permissive temperature. These properties were interpreted as providing genetic evidence for interaction of the dnaE and dnaN gene products in E. coli. Images PMID:6458043

  18. Functional cooperation of the dnaE and dnaN gene products in Escherichia coli.

    PubMed

    Kuwabara, N; Uchida, H

    1981-09-01

    A system was designed to isolate second-site intergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the alpha subunit of DNA polymerase III [McHenry, C. S. & Crow, W. (1979) J. Biol. Chem. 254, 1748-1753]. One such suppressor, named sueA77, was finely mapped and found to be located at 82 min on the E. coli chromosome, between dnaA and recF, and within the dnaN gene [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553]. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme [Burgers, P. M. J., Kornberg, A. & Sakakibara, Y. (1981) Proc. Natl. Acad. Sci. USA 78, 5391-5395]. The sueA77 mutation was trans-dominant over its wild-type allele, and it suppressed different thermosensitive mutations of dnaE with different maximal permissive temperature. These properties were interpreted as providing genetic evidence for interaction of the dnaE and dnaN gene products in E. coli.

  19. The transport of antibiotic resistance genes and residues in groundwater near swine production facilities

    NASA Astrophysics Data System (ADS)

    Lin, Y. F.; Yannarell, A. C.; Mackie, R. I.; Krapac, I. G.; Chee-Sanford, J. S.; Koike, S.

    2008-12-01

    The use of antibiotics at concentrated animal feeding operations (CAFOs) for disease prevention, disease treatment, and growth promotion can contribute to the spread of antibiotic compounds, their breakdown products, and antibiotic resistant bacteria and/or the genes that confer resistance. In addition, constitutive use of antibiotics at sub-therapeutic levels can select for antibiotic resistance among the bacteria that inhabit animal intestinal tracts, onsite manure treatment facilities, and any environments receiving significant inputs of manure (e.g. through waste lagoon leakage or fertilizer amendments to farm soils). If the antibiotic resistant organisms persist in these new environments, or if they participate in genetic exchanges with the native microflora, then CAFOs may constitute a significant reservoir for the spread of antibiotic resistance to the environment at large. Our results have demonstrated that leakage from waste treatment lagoons can influence the presence and persistence of tetracycline resistance genes in the shallow aquifer adjacent to swine CAFOs, and molecular phylogeny allowed us to distinguish "native" tetracycline resistance genes in control groundwater wells from manure-associated genes introduced from the lagoon. We have also been able to detect the presence of erythromycin resistance genes in CAFO surface and groundwater even though erythromycin is strictly reserved for use in humans and thus is not utilized at any of these sites. Ongoing research, including modeling of particle transport in groundwater, will help to determine the potential spatial and temporal extent of CAFO-derived antibiotic resistance.

  20. Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes.

    PubMed

    Staack, Roland F; Paul, Liane D; Springer, Dietmar; Kraemer, Thomas; Maurer, Hans H

    2004-01-15

    1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.

  1. Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum.

    PubMed

    Douma, Rutger D; Verheijen, Peter J T; de Laat, Wim T A M; Heijnen, Joseph J; van Gulik, Walter M

    2010-07-01

    As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q(p)) and growth rate (mu) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q(p)(mu) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed-batch experiments, leads to errors in the prediction, because q(p) is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate-limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin-N synthase (IPNS) was the main rate-limiting enzyme for penicillin-G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady-state as well as the dynamics during chemostat start-up and fed-batch cultivation.

  2. Engineering cell lines for production of replication defective HSV-1 gene therapy vectors.

    PubMed

    Grant, Kyle G; Krisky, David M; Ataai, Mohammed M; Glorioso, Joseph C

    2009-03-01

    Herpes simplex virus type 1 (HSV-1) represents an attractive vehicle for a variety of gene therapy applications. To render this virus safe for clinical use, its cytotoxic genes must be removed without losing its ability to express transgenes efficiently. Our vectors are deleted for the essential immediate early genes ICP4 and ICP27. These genes are controlled by unique promoters having enhancer elements responsive to a viral structural protein VP16. The expression of these genes occurs prior to the activation of all other lytic functions and is thus required to initiate and complete the virus replication cycle. For large scale manufacture of clinical grade vectors, efficient cell lines must be generated that express the essential viral gene products in trans during vector propagation. Here we describe methods for engineering HSV-1 production cell lines that improve vector growth by altering the kinetics of complementing gene expression. We examined the ability of Vero cells independently transduced with ICP4 and ICP27 under transcriptional control of their respective promoters to support the growth of a replication defective vector (JDTOZHE), deleted for ICP4, ICP27 and approximately 20 kb of internal elements that are not required for virus growth in Vero cells. Vector yield on this cell line was 3 logs lower than wild-type virus grown on Vero cells. To understand the mechanism underlying poor vector yield, we examined the expression of ICP4 and ICP27 during virus complementation. While ICP27 was expressed immediately on vector infection, the expression of ICP4 was considerably delayed by 8-10 h, suggesting that the ICP4 promoter was not adequately activated by VP16 delivered by the infectious vector particle. Use of the ICP0 promoter to express ICP4 from the cellular genome resulted in higher induction levels and faster kinetics of ICP4 expression and a 10-fold improvement in vector yield. This study suggests that vector complementation is highly dependent on the

  3. Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

    USGS Publications Warehouse

    Mackie, R.I.; Koike, S.; Krapac, I.; Chee-Sanford, J.; Maxwell, Susan; Aminov, R.I.

    2006-01-01

    Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment.To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators-including inorganic ions, antibiotics, and antibiotic resistance genes-were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 ??g/L.Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and

  4. Secreted proteins and genes in fetal and neonatal pig adipose tissue and stromal-vascular cells.

    PubMed

    Hausman, G J; Poulos, S P; Richardson, R L; Barb, C R; Andacht, T; Kirk, H C; Mynatt, R L

    2006-07-01

    Although microarray and proteomic studies have indicated the expression of unique and unexpected genes and their products in human and rodent adipose tissue, similar studies of meat animal adipose tissue have not been reported. Thus, total RNA was isolated from stromal-vascular (S-V) cell cultures (n = 4; 2 arrays; 2 cultures/array) from 90-d (79% of gestation) fetuses and adipose tissue from 105-d (92% of gestation) fetuses (n = 2) and neonatal (5-d-old) pigs (n = 2). Duplicate adipose tissue microarrays (n = 4) represented RNA samples from a pig and a fetus. Dye-labeled cDNA probes were hybridized to custom microarrays (70-mer oligonucleotides) representing more than 600 pig genes involved in growth and reproduction. Microarray studies showed significant expression of 40 genes encoding for known adipose tissue secreted proteins in fetal S-V cell cultures and adipose tissue. Expression of 10 genes encoding secreted proteins not known to be expressed by adipose tissue was also observed in neonatal adipose tissue and fetal S-V cell cultures. Additionally, the agouti gene was detected by reverse transcription-PCR in pig S-V cultures and adipose tissue. Proteomic analysis of adipose tissue and fetal and young pig S-V cell culture-conditioned media identified multiple secreted proteins including heparin-like epidermal growth factor-like growth factor and several apolipoproteins. Another adipose tissue secreted protein, plasminogen activator inhibitor-1, was identified by ELISA in S-V cell culture media. A group of 20 adipose tissue secreted proteins were detected or identified using the gene microarray and the proteomic and protein assay approaches including apolipoprotein-A1, apolipoprotein-E, relaxin, brain-derived neurotrophic factor, and IGF binding protein-5. These studies demonstrate, for the first time, the expression of several major secreted proteins in pig adipose tissue that may influence local and central metabolism and growth.

  5. Production of Phloroglucinol, a Platform Chemical, in Arabidopsis using a Bacterial Gene

    PubMed Central

    Abdel-Ghany, Salah E.; Day, Irene; Heuberger, Adam L.; Broeckling, Corey D.; Reddy, Anireddy S.N.

    2016-01-01

    Phloroglucinol (1,3,5-trihydroxybenzene; PG) and its derivatives are phenolic compounds that are used for various industrial applications. Current methods to synthesize PG are not sustainable due to the requirement for carbon-based precursors and co-production of toxic byproducts. Here, we describe a more sustainable production of PG using plants expressing a native bacterial or a codon-optimized synthetic PhlD targeted to either the cytosol or chloroplasts. Transgenic lines were analyzed for the production of PG using gas and liquid chromatography coupled to mass spectroscopy. Phloroglucinol was produced in all transgenic lines and the line with the highest PhlD transcript level showed the most accumulation of PG. Over 80% of the produced PG was glycosylated to phlorin. Arabidopsis leaves have the machinery to glycosylate PG to form phlorin, which can be hydrolyzed enzymatically to produce PG. Furthermore, the metabolic profile of plants with PhlD in either the cytosol or chloroplasts was altered. Our results provide evidence that plants can be engineered to produce PG using a bacterial gene. Phytoproduction of PG using a bacterial gene paves the way for further genetic manipulations to enhance the level of PG with implications for the commercial production of this important platform chemical in plants. PMID:27924918

  6. Manipulation of IL-10 gene expression by Toxoplasma gondii and its products

    PubMed Central

    Pestechian, Nader; Khanahmad Shahreza, Hosein; Faridnia, Roghiyeh; Kalani, Hamed

    2016-01-01

    Background: This study was designed to evaluate whether or not T. gondii and its derivatives can change the gene expression level of IL-10 in murine leukocytes in vivo. Methods: Fifty BALB/c mice were divided into 5 groups, four of which received the excretory/secretory product (ESP) from cell culture medium, the ESP from cell free medium, the Toxoplasma lysate product (TLP) and the active tachyzoites, respectively. The fifth group was considered as control and received PBS. The peritoneal leukocytes from the mice were collected. Their total RNA were extracted and converted to cDNA and the gene expression levels of IL-10 in the samples were evaluated by quantitative real time-PCR using the REST-2009 software. Results: The findings showed a decrease in the expression level of IL-10 in the TLP group (p=0.004). Moreover, the IL-10 gene expression level was upregulated in the group of the ESP from cell culture medium (p=0.04) and the active tachyzoite group (p=0.04). The expression of IL-10 gene in the group of ESP from cell-free medium was not significant compared to the control one (p=0.45). Conclusion: T. gondii and its derivatives are able to increase (the active T. gondii tachyzoite and the ESP from cell culture medium) and decrease (the TLP) the gene expression level of IL-10 in a murine model. The question remains to be examined in further study about which molecules are involved in this process. PMID:27683651

  7. Cloning and functional analysis by gene disruption of a novel gene involved in indigo production and fluoranthene metabolism in Pseudomonas alcaligenes PA-10.

    PubMed

    Alemayehu, D; Gordon, L M; O'Mahony, M M; O'Leary, N D; Dobson, A D W

    2004-10-15

    A novel indole dioxygenase (idoA) gene has been cloned from Pseudomonas alcaligenes PA-10, based on its ability to convert indole to indigo. The chromosomally encoded idoA gene exhibits no similarity to previously cloned naphthalene dioxygenases or to aromatic oxygenases from other species at the nucleotide level. Phylogenetic analysis indicates that the idoA gene product is most similar to an acyl-CoA dehydrogenase from Novosphingobium aromaticivorans. The enzyme encoded by the idoA gene is essential for the metabolism of fluoranthene, since a mutant in which the idoA gene has been disrupted looses the ability to degrade this compound. The idoA gene appears to be constitutively expressed in PA-10, but its expression is also subject to regulation following prior exposure to salicylate and to fluoranthene degradative intermediates.

  8. Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis.

    PubMed

    Doruk, Tugrul; Avican, Ummehan; Camci, Irem Yalim; Gedik, Sedef Tunca

    2013-05-06

    Polyphosphate (polyP), synthesized by polyP kinase (PPK) using the terminal phosphate of ATP as substrate, performs important functions in every living cell. The present work reports on the relationship between polyP metabolism and bioinsecticide production in Bacillus thuringiensis subsp. israelensis (Bti). The ppk gene of Bti was cloned into vector pHT315 and the effect of its overexpression on endotoxin production was determined. Endotoxin production by the recombinant strain was found to be consistently higher than that by the wild type strain and the strain that carried the empty plasmid. The toxicity of the recombinant mutant strain (LC50 5.8±0.6ngml(-1)) against late 2nd instar Culex quinquefasciatus was about 7.7 times higher than that of Bti (LC50 44.9±7ngml(-1)). To our knowledge this is the first reported study which relates polyP metabolism with bioinsecticide biosynthesis.

  9. Engineering Klebsiella oxytoca for efficient 2, 3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene.

    PubMed

    Ji, Xiao-Jun; Huang, He; Zhu, Jian-Guo; Ren, Lu-Jing; Nie, Zhi-Kui; Du, Jun; Li, Shuang

    2010-02-01

    Ethanol was a major byproduct of 2,3-butanediol (2,3-BD) fermentation by Klebsiella oxytoca ME-UD-3. In order to achieve a high efficiency of 2,3-BD production, K. oxytoca mutants deficient in ethanol formation were successfully constructed by replace the aldA gene coding for aldehyde dehydrogenase with a tetracycline resistance cassette. The results suggested that inactivation of aldA led to a significantly improved 2,3-BD production. The carbon flux to 2,3-BD was enhanced by eliminating the byproducing ethanol and at the same time reducing the accumulation of another byproduct acetoin. At last, by fed-batch culturing of the mutant, the final 2,3-BD titer up to 130 g/l with the productivity of 1.63 g/l.h and the 2,3-BD yield relative to glucose of 0.48 g/g was obtained.

  10. Prevalence of ten putative virulence genes in the emerging foodborne pathogen Arcobacter isolated from food products.

    PubMed

    Girbau, Cecilia; Guerra, Cristian; Martínez-Malaxetxebarria, Irati; Alonso, Rodrigo; Fernández-Astorga, Aurora

    2015-12-01

    Arcobacter spp. are considered to be emerging food- and waterborne pathogens for both humans and animals. However, their virulence mechanisms are still poorly understood. In this study the presence of ten virulence genes (cadF, ciaB, cj1349, hecA, hecB, mviN, pldA, irgA, tlyA and iroE) was assessed in a set of 47 strains of Arcobacter butzleri, 10 of Arcobacter cryaerophilus and 1 Arcobacter skirrowii strain recovered from different food products (pork, chicken, beef, milk, clams and mussels). Overall, the genes cadF, ciaB, cj1349, mviN, pldA and tlyA were detected in all A. butzleri and A. skirrowii strains. Lower detection rates were observed for irgA, iroE, hecA and hecB. The genes hecB and iroE were detected neither in A. cryaerophilus nor in A. skirrowii. The genes hecA and irgA were not detected in A. skirrowii. It was noteworthy that the genes hecA and hecB were significantly (P < 0.05) highly detected in A. butzleri strains isolated from clams compared with strains isolated from milk and chicken. Therefore, our findings underline clams as a source of A. butzleri strains with high prevalence of putative virulence genes. This could be hazardous to human health, especially because these bivalves are usually consumed raw or undercooked.

  11. Silver Resistance Genes Are Overrepresented among Escherichia coli Isolates with CTX-M Production

    PubMed Central

    Edquist, Petra; Sandegren, Linus; Adler, Marlen; Tängdén, Thomas; Drobni, Mirva; Olsen, Björn; Melhus, Åsa

    2014-01-01

    Members of the Enterobacteriaceae with extended-spectrum beta-lactamases (ESBLs) of the CTX-M type have disseminated rapidly in recent years and have become a threat to public health. In parallel with the CTX-M type expansion, the consumption and widespread use of silver-containing products has increased. To determine the carriage rates of silver resistance genes in different Escherichia coli populations, the presence of three silver resistance genes (silE, silP, and silS) and genes encoding CTX-M-, TEM-, and SHV-type enzymes were explored in E. coli isolates of human (n = 105) and avian (n = 111) origin. The antibiotic profiles were also determined. Isolates harboring CTX-M genes were further characterized, and phenotypic silver resistance was examined. The silE gene was present in 13 of the isolates. All of them were of human origin. Eleven of these isolates harbored ESBLs of the CTX-M type (P = 0.007), and eight of them were typed as CTX-M-15 and three as CTX-M-14. None of the silE-positive isolates was related to the O25b-ST131 clone, but 10 out of 13 belonged to the ST10 or ST58 complexes. Phenotypic silver resistance (silver nitrate MIC > 512 mg/liter) was observed after silver exposure in 12 of them, and a concomitant reduced susceptibility to piperacillin-tazobactam developed in three. In conclusion, 12% of the human E. coli isolates but none of the avian isolates harbored silver resistance genes. This indicates another route for or level of silver exposure for humans than that caused by common environmental contamination. Since silE-positive isolates were significantly more often found in CTX-M-positive isolates, it is possible that silver may exert a selective pressure on CTX-M-producing E. coli isolates. PMID:25128339

  12. Novel diabetes mellitus treatment: mature canine insulin production by canine striated muscle through gene therapy.

    PubMed

    Niessen, S J M; Fernandez-Fuente, M; Mahmoud, A; Campbell, S C; Aldibbiat, A; Huggins, C; Brown, A E; Holder, A; Piercy, R J; Catchpole, B; Shaw, J A M; Church, D B

    2012-07-01

    Muscle-targeted gene therapy using insulin genes has the potential to provide an inexpensive, low maintenance alternative or adjunctive treatment method for canine diabetes mellitus. A canine skeletal muscle cell line was established through primary culture, as well as through transdifferentiation of canine fibroblasts after infection with a myo-differentiation gene containing adenovirus vector. A novel mutant furin-cleavable canine preproinsulin gene insert (cppI4) was designed and created through de novo gene synthesis. Various cell lines, including the generated canine muscle cell line, were transfected with nonviral plasmids containing cppI4. Insulin and desmin immunostaining were used to prove insulin production by muscle cells and specific canine insulin ELISA to prove mature insulin secretion into the medium. The canine myoblast cultures proved positive on desmin immunostaining. All cells tolerated transfection with cppI4-containing plasmid, and double immunostaining for insulin and desmin proved present in the canine cells. Canine insulin ELISA assessment of medium of cppI4-transfected murine myoblasts and canine myoblast and fibroblast mixture proved presence of mature fully processed canine insulin, 24 and 48 h after transfection. The present study provides proof of principle that canine muscle cells can be induced to produce and secrete canine insulin on transfection with nonviral plasmid DNA containing a novel mutant canine preproinsulin gene that produces furin-cleavable canine preproinsulin. This technology could be developed to provide an alternative canine diabetes mellitus treatment option or to provide a constant source for background insulin, as well as C-peptide, alongside current treatment options.

  13. Silver resistance genes are overrepresented among Escherichia coli isolates with CTX-M production.

    PubMed

    Sütterlin, Susanne; Edquist, Petra; Sandegren, Linus; Adler, Marlen; Tängdén, Thomas; Drobni, Mirva; Olsen, Björn; Melhus, Asa

    2014-11-01

    Members of the Enterobacteriaceae with extended-spectrum beta-lactamases (ESBLs) of the CTX-M type have disseminated rapidly in recent years and have become a threat to public health. In parallel with the CTX-M type expansion, the consumption and widespread use of silver-containing products has increased. To determine the carriage rates of silver resistance genes in different Escherichia coli populations, the presence of three silver resistance genes (silE, silP, and silS) and genes encoding CTX-M-, TEM-, and SHV-type enzymes were explored in E. coli isolates of human (n = 105) and avian (n = 111) origin. The antibiotic profiles were also determined. Isolates harboring CTX-M genes were further characterized, and phenotypic silver resistance was examined. The silE gene was present in 13 of the isolates. All of them were of human origin. Eleven of these isolates harbored ESBLs of the CTX-M type (P = 0.007), and eight of them were typed as CTX-M-15 and three as CTX-M-14. None of the silE-positive isolates was related to the O25b-ST131 clone, but 10 out of 13 belonged to the ST10 or ST58 complexes. Phenotypic silver resistance (silver nitrate MIC > 512 mg/liter) was observed after silver exposure in 12 of them, and a concomitant reduced susceptibility to piperacillin-tazobactam developed in three. In conclusion, 12% of the human E. coli isolates but none of the avian isolates harbored silver resistance genes. This indicates another route for or level of silver exposure for humans than that caused by common environmental contamination. Since silE-positive isolates were significantly more often found in CTX-M-positive isolates, it is possible that silver may exert a selective pressure on CTX-M-producing E. coli isolates.

  14. Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system.

    PubMed

    Zhang, Dawei; Shrestha, Binaya; Li, Zhaopeng; Tan, Tianwei

    2007-01-01

    Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ- 10 production, we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-beta-D-thiogalactopyranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.

  15. Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production.

    PubMed

    Cho, Kwang-Soo; Han, Eun-Heui; Kwak, Sang-Soo; Cho, Ji-Hong; Im, Ju-Seong; Hong, Su-Young; Sohn, Hwang-Bae; Kim, Yun-Hee; Lee, Shin-Woo

    2016-01-01

    Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits.

  16. Nucleic acid binding property of the gene products of rice stripe virus.

    PubMed

    Liang, Delin; Ma, Xiangqiang; Qu, Zhicai; Hull, Roger

    2005-10-01

    GST fusion proteins of the six gene products from RNAs 2,3 and 4 of the tenuivirus, Rice stripe virus (RSV), were used to study the nucleic acid binding activities in vitro. Three of the proteins, p3, pc3 and pc4, bound both single- and double-stranded cDNA of RSV RNA4 and also RNA3 transcribed from its cDNA clone, while p2, pc2-N (the N-terminal part of pc2) nor p4 bound the cDNA or RNA transcript. The binding activity of p3 is located in the carboxyl-terminus amino acid 154-194, which contains basic amino acid rich beta-sheets. The acidic amino acid-rich amino-terminus (amino acids 1-100) of p3 did not have nucleic acid binding activity. The related analogous gene product of the tenuivirus, Rice hoja blanca virus, is a suppressor of gene silencing and the possibility of the nucleic acid binding ability of RSV p3 being associated with this property is discussed. The C-terminal part of the RSV nucleocapsid protein, which also contains a basic region, binds nucleic acids, which is consistent with its function. The central and C-terminal regions of pc4 bind nucleic acid. It has been suggested that this protein is a cell-to-cell movement protein and nucleic acid binding would be in accord with this function.

  17. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

    PubMed

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-03-09

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

  18. Identification and characterization of a novel yeast gene: the YGP1 gene product is a highly glycosylated secreted protein that is synthesized in response to nutrient limitation.

    PubMed Central

    Destruelle, M; Holzer, H; Klionsky, D J

    1994-01-01

    Nutrient starvation in the yeast Saccharomyces cerevisiae leads to a number of physiological changes that accompany entry into stationary phase. The expression of genes whose products play a role in stress adaptation is regulated in a manner that allows the cell to sense and respond to changing environmental conditions. We have identified a novel yeast gene, YGP1, that displays homology to the sporulation-specific SPS100 gene. The expression of YGP1 is regulated by nutrient availability. The gene is expressed at a basal level during "respiro-fermentative" (logarithmic) growth. When the glucose concentration in the medium falls below 1%, the YGP1 gene is derepressed and the gene product, gp37, is synthesized at levels up to 50-fold above the basal level. The glucose-sensing mechanism is independent of the SNF1 pathway and does not operate when cells are directly shifted to a low glucose concentration. The expression of YGP1 also responds to the depletion of nitrogen and phosphate, indicating a general response to nutrient deprivation. These results suggest that the YGP1 gene product may be involved in cellular adaptations prior to stationary phase and may be a useful marker protein for monitoring early events associated with the stress response. Images PMID:8139573

  19. Primary structure of the Escherichia coli thyA gene and its thymidylate synthase product.

    PubMed Central

    Belfort, M; Maley, G; Pedersen-Lane, J; Maley, F

    1983-01-01

    The nucleotide sequence of a 1,163-base-pair fragment that encodes the entire thyA gene of Escherichia coli K-12 was determined. The strategy involved sequence determination of both DNA strands by using overlapping deletions that had been generated in vitro from the two ends of the fragment with BAL-31 nuclease. The amino-terminal sequence of thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45), the product of the thyA gene, located the 792-base-pair open reading frame, which codes for the 264 amino acid residues of this enzyme. The amino acid sequence deduced from the nucleotide data was confirmed to the extent of 40% by partial sequence analysis of the enzyme purified from extracts of the amplified cloned gene. Transcriptional and translational control areas were apparent in the regions flanking the structural gene. The 5-fluorodeoxyuridylate-binding residue of the active site was identified as cysteine-146. Comparison of the E. coli and Lactobacillus casei synthase sequences reveals consistent homology (62%) over extensive regions. This homology is particularly striking in a very hydrophobic region bordering cysteine-146. In the two enzymes, this region, which probably defines the active site, is 82% homologous. However, a dramatic difference between the two sequences is reflected by the surprising finding that a 51-amino-acid stretch, present midway through the L. casei sequence, is completely absent from the E. coli enzyme. PMID:6308660

  20. Dissection of Maize Kernel Composition and Starch Production by Candidate Gene Association

    PubMed Central

    Wilson, Larissa M.; Whitt, Sherry R.; Ibáñez, Ana M.; Rocheford, Torbert R.; Goodman, Major M.; Buckler, Edward S.

    2004-01-01

    Cereal starch production forms the basis of subsistence for much of the world's human and domesticated animal populations. Starch concentration and composition in the maize (Zea mays ssp mays) kernel are complex traits controlled by many genes. In this study, an association approach was used to evaluate six maize candidate genes involved in kernel starch biosynthesis: amylose extender1 (ae1), brittle endosperm2 (bt2), shrunken1 (sh1), sh2, sugary1, and waxy1. Major kernel composition traits, such as protein, oil, and starch concentration, were assessed as well as important starch composition quality traits, including pasting properties and amylose levels. Overall, bt2, sh1, and sh2 showed significant associations for kernel composition traits, whereas ae1 and sh2 showed significant associations for starch pasting properties. ae1 and sh1 both associated with amylose levels. Additionally, haplotype analysis of sh2 suggested this gene is involved in starch viscosity properties and amylose content. Despite starch concentration being only moderately heritable for this particular panel of diverse maize inbreds, high resolution was achieved when evaluating these starch candidate genes, and diverse alleles for breeding and further molecular analysis were identified. PMID:15377761

  1. Metabolic engineering of Escherichia coli for ethanol production without foreign genes

    NASA Astrophysics Data System (ADS)

    Kim, Youngnyun

    Worldwide dependence on finite petroleum-based energy necessitates alternative energy sources that can be produced from renewable resources. A successful example of an alternative transportation fuel is bioethanol, produced by microorganisms, from corn starch that is blended with gasoline. However, corn, currently the main feedstock for bioethanol production, also occupies a significant role in human food and animal feed chains. As more corn is diverted to bioethanol, the cost of corn is expected to increase with an increase in the price of food, feed and ethanol. Using lignocellulosic biomass for ethanol production is considered to resolve this problem. However, this requires a microbial biocatalyst that can ferment hexoses and pentoses to ethanol. Escherichia coli is an efficient biocatalyst that can use all the monomeric sugars in lignocellulose, and recombinant derivatives of E. coli have been engineered to produce ethanol as the major fermentation product. In my study, ethanologenic E. coli strains were isolated from a ldhA-, pflB- derivative without introduction of foreign genes. These isolates grew anaerobically and produced ethanol as the main fermentation product. The mutation responsible for anaerobic growth and ethanol production was mapped in the lpdA gene and the mutation was identified as E354K in three of the isolates tested. Another three isolates carried an lpdA mutation, H352Y. Enzyme kinetic studies revealed that the mutated form of the dihydrolipoamide dehydrogenase (LPD) encoded by the lpdA was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity to NADH of the pyruvate dehydrogenase complex in strain SE2378. The net yield of 4 moles of NADH and 2 moles of acetyl-CoA per mole of glucose produced by a combination of glycolysis and PDH provided a logical basis to explain the production of 2 moles of ethanol per glucose. The development of E

  2. Identification of a putative FR901469 biosynthesis gene cluster in fungal sp. No. 11243 and enhancement of the productivity by overexpressing the transcription factor gene frbF.

    PubMed

    Matsui, Makoto; Yokoyama, Tatsuya; Nemoto, Kaoru; Kumagai, Toshitaka; Terai, Goro; Tamano, Koichi; Machida, Masayuki; Shibata, Takashi

    2017-02-01

    FR901469 is an antifungal antibiotic produced by fungal sp. No. 11243. Here, we searched for FR901469 biosynthesis genes in the genome of No. 11243. Based on the molecular structure of FR901469 and endogenous functional motifs predicted in each genomic NRPS gene, a putative FR901469 biosynthesis gene cluster harboring the most plausible NRPS gene was identified. A transcription factor gene, designated frbF, was found in the cluster. To improve FR901469 productivity, we constructed a strain in which frbF was overexpressed and named it TFH2-2. FR901469 productivity of TFH2-2 was 3.4 times higher than that of the wild-type strain. Transcriptome analysis revealed that most of the genes in the putative FR901469 biosynthesis gene cluster were upregulated in TFH2-2. It also showed that the expression of genes related to ergosterol biosynthesis, β-1,3-glucan catabolism, and chitin synthesis was inclined to exhibit significant differences in TFH2-2.

  3. Production of penicillin by fungi growing on food products: identification of a complete penicillin gene cluster in Penicillium griseofulvum and a truncated cluster in Penicillium verrucosum.

    PubMed

    Laich, Federico; Fierro, Francisco; Martín, Juan F

    2002-03-01

    Mycobiota growing on food is often beneficial for the ripening and development of the specific flavor characteristics of the product, but it can also be harmful due to the production of undesirable compounds such as mycotoxins or antibiotics. Some of the fungi most frequently isolated from fermented and cured meat products such as Penicillium chrysogenum and Penicillium nalgiovense are known penicillin producers; the latter has been shown to be able to produce penicillin when growing on the surface of meat products and secrete it to the medium. The presence of penicillin in food must be avoided, since it can lead to allergic reactions and the arising of penicillin resistance in human-pathogenic bacteria. In this article we describe a study of the penicillin production ability among fungi of the genus Penicillium that are used as starters for cheese and meat products or that are frequently isolated from food products. Penicillium griseofulvum was found to be a new penicillin producer and to have a penicillin gene cluster similar to that of Penicillium chrysogenum. No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P. verrucosum, which contains the pcbAB gene (as shown by hybridization and PCR cloning of fragments of the gene) but lacks pcbC and penDE. Antibacterial activities due to the production of secondary metabolites other than penicillin were observed in some fungi.

  4. Deletion of pigR gene in Monascus ruber leads to loss of pigment production.

    PubMed

    Xie, Nana; Liu, Qingpei; Chen, Fusheng

    2013-09-01

    Pigments produced by Monascus are traditional food colorants and are widely used as dietary supplements. Since genes involving in pigment biosynthesis have not been reported, we describe the identification of a putative pigment-regulatory gene (pigR) obtained by molecular analysis of an albino strain of Monascus ruber M7. In the pigR-deleted strain (ΔpigR), neither the pigments nor pigR expression were detected by HPLC or reverse-transcription PCR, respectively, whereas the introduction of the pigR, together with a constitutive trpC promoter into ΔpigR, caused it to produce 5.4 U of red pigments/g dry mycelia, about 12-fold higher than Monascus ruber M7 (0.46 U/g dry mycelia). Thus pigR up-regulates pigment production in Monascus ruber M7.

  5. Identification of the human papilloma virus-1a E4 gene products.

    PubMed Central

    Doorbar, J; Campbell, D; Grand, R J; Gallimore, P H

    1986-01-01

    Antibodies prepared against a human papilloma virus-1 (HPV-1) E4/beta-galactosidase fusion protein identified several polypeptides in HPV-1, but not HPV-2 or 4, induced papillomas. The major E4 protein, that represented up to 30% of total cellular protein, was a 16/17-K doublet which was purified by column chromatography and analysed for amino acid content. A peptide derived by chymotryptic digestion was purified by h.p.l.c. and subjected to amino acid sequencing. The unique sequence obtained, Gly-His-Pro-Asp-Leu-Ser-Leu, identified the 16/17-K doublet as a product of the HPV-1 E4 gene region. Antibodies to both the E4/beta-galactosidase fusion protein and the 16/17-K doublet identified two smaller polypeptides (10/11-K) which may represent spliced products of E4. We propose that the products of the HPV-1 E4 gene region are not classical DNA tumor virus early proteins and suggest that they play a role in virus maturation. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3011404

  6. Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis

    PubMed Central

    Westers, Helga; Braun, Peter G.; Westers, Lidia; Antelmann, Haike; Hecker, Michael; Jongbloed, Jan D. H.; Yoshikawa, Hirofumi; Tanaka, Teruo; van Dijl, Jan Maarten; Quax, Wim J.

    2005-01-01

    Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor. PMID:15812018

  7. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

    2015-03-01

    Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

  8. Genetic association between SNPs in the DGAT1 gene and milk production traits in Murrah buffaloes.

    PubMed

    de Freitas, Ana Cláudia; de Camargo, Gregório Miguel Ferreira; Stafuzza, Nedenia Bonvino; Aspilcueta-Borquis, Rusbel Raul; Venturini, Guilherme Costa; Dias, Marina Mortati; Cardoso, Diercles Francisco; Tonhati, Humberto

    2016-10-01

    This study identified polymorphisms in the DGAT1 gene in Murrah buffaloes and investigated the associations to milk production and quality traits (milk, fat and protein yields and percentages, somatic cell count). Genomic DNA was extracted from hair follicles collected from the tail of 196 females. Three SNPs were identified in DGAT1 gene by sequencing. Statistical analyses were performed to verify the linkage and the association between polymorphisms and traits. The estimated value of r (2) between two SNPs in exon 17 (g.11,783G > A and g.11,785 T > C) was 0.029. SNP g.11,785 T > C was significantly associated (P < 0.05) to fat and protein percentage. Dominance effect was significant for milk and fat yields and protein percentage (P < 0.05). The additive effect of the SNP g.11,785 T > C was significant for protein production and somatic cell count (P < 0.05). It indicates that assisted marker selection might be done with considerations to balance production and udder health.

  9. Increased biomass production of industrial bakers' yeasts by overexpression of Hap4 gene.

    PubMed

    Dueñas-Sánchez, Rafael; Codón, Antonio C; Rincón, Ana M; Benítez, Tahía

    2010-10-15

    HAP4 encodes a transcriptional activator of respiration-related genes and so, redirection from fermentation to respiration flux should give rise to an increase in biomass production in Saccharomyces cerevisiae transformants that overexpress HAP4. With this aim, three bakers' yeasts, that is, V1 used for lean doughs, its 2-deoxy-D-glucose resistant derivative DOG21, and V3 employed for sweet doughs, were transformed with integrative cassettes that carried HAP4 gene under the control of constitutive promoter pTEF2; in addition VTH, DTH and 3TH transformants were selected and characterized. Transformants showed increased expression of HAP4 and respiration-related genes such as QCR7 and QCR8 with regard to parental, and similar expression of SUC2 and MAL12; these genes are relevant in bakers' industry. Invertase (Suc2p) and maltase (Mal12p) activities, growth and sugar consumption rates in laboratory (YPD) or industrial media (MAB) were also comparable in bakers' strains and their transformants, but VTH, DTH and 3TH increased their final biomass production by 9.5, 5.0 and 5.0% respectively as compared to their parentals in MAB. Furthermore, V1 and its transformant VTH had comparable capacity to ferment lean doughs (volume increase rate and final volume) while V3 and its transformant 3TH fermented sweet doughs in a similar manner. Therefore transformants possessed increased biomass yield and appropriate characteristics to be employed in bakers' industry because they lacked drug resistant markers and bacterial DNA, and were genetically stable.

  10. Gut symbiotic bacteria stimulate insect growth and egg production by modulating hexamerin and vitellogenin gene expression.

    PubMed

    Lee, Jun Beom; Park, Kyoung-Eun; Lee, Seung Ah; Jang, Seong Han; Eo, Ho Jeong; Jang, Ho Am; Kim, Chan-Hee; Ohbayashi, Tsubasa; Matsuura, Yu; Kikuchi, Yoshitomo; Futahashi, Ryo; Fukatsu, Takema; Lee, Bok Luel

    2017-04-01

    Recent studies have suggested that gut symbionts modulate insect development and reproduction. However, the mechanisms by which gut symbionts modulate host physiologies and the molecules involved in these changes are unclear. To address these questions, we prepared three different groups of the insect Riptortus pedestris: Burkholderia gut symbiont-colonized (Sym) insects, Burkholderia-non-colonized (Apo) insects, and Burkholderia-depleted (Sym(Burk-)) insects, which were fed tetracycline. When the hemolymph proteins of three insects were analyzed by SDS-PAGE, the hexamerin-α, hexamerin-β and vitellogenin-1 proteins of Sym-adults were highly expressed compared to those of Apo- and Sym(Burk-)-insects. To investigate the expression patterns of these three genes during insect development, we measured the transcriptional levels of these genes. The hexamerin-β gene was specifically expressed at all nymphal stages, and its expression was detected 4-5 days earlier in Sym-insect nymphs than that in Apo- and Sym(Burk-)-insects. However, the hexamerin-α and vitellogenin-1 genes were only expressed in adult females, and they were also detected 6-7 days earlier and were 2-fold higher in Sym-adult females than those in the other insects. Depletion of hexamerin-β by RNA interference in 2nd instar Sym-nymphs delayed adult emergence, whereas hexamerin-α and vitellogenin-1 RNA interference in 5th instar nymphs caused loss of color of the eggs of Sym-insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production by regulating production of these three hemolymph storage proteins.

  11. Identification of genes affecting production of the adhesion organelle of Caulobacter crescentus CB2.

    PubMed Central

    Mitchell, D; Smit, J

    1990-01-01

    Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2. A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis. Ten unique sites of Tn5 insertion affecting holdfast function were identified that were clustered in four regions of the genome. Representative mutants of the 10 Tn5 insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness. Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell. With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants. This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures. Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production. Images PMID:2168382

  12. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins

    SciTech Connect

    Strebel, K.; Beck, E.; Strohmaier, K.; Schaller, H.

    1986-03-01

    Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible lambdaPL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in /sup 35/S-labeled extracts from foot-and-mouth disease virus-infected BHK cells. This allowed us to locate unequivocally all mature foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and mouth disease virus gene products not previously identified in vivo or in vitro.

  13. Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae

    DOEpatents

    Melis, Anastasios; Mitra, Mautusi

    2010-06-29

    The invention provides method and compositions to minimize the chlorophyll antenna size of photosynthesis by decreasing TLA1 gene expression, thereby improving solar conversion efficiencies and photosynthetic productivity in plants, e.g., green microalgae, under bright sunlight conditions.

  14. Black agouti (ACI) rats show greater drug- and cue-induced reinstatement of methamphetamine-seeking behavior than Fischer 344 and Lewis rats.

    PubMed

    Xi, Jinlei; Kruzich, Paul J

    2007-05-01

    Fischer 344 (F344) and Lewis (LEW) rats differ in methamphetamine self-administration (SA) and methamphetamine-induced reinstatement of previously extinguished behavior. We sought to determine whether genetic background also influences methamphetamine reinforcement efficacy, conditioned reinstatement, and methamphetamine-primed reinstatement of responding in F344, LEW, and Black Agouti (ACI) rats. We implanted rats with jugular catheters and trained them to self-administer methamphetamine (0.06 mg/kg/infusion) under a progressive ratio (PR) schedule of reinforcement during daily 2-h SA sessions. A compound stimulus (light+tone; LT) was paired with each infusion. Dose-dependent intake was determined for each rat. Rats then entered the extinction phase of the experiment where responding resulted in no programmed consequences. Following extinction sessions, rats underwent conditioned reinstatement testing. For conditioned reinstatement, rats received response-contingent presentations of the LT and no methamphetamine. Last, methamphetamine-primed reinstatement test sessions where conducted where subjects received experimenter delivered infusions of methamphetamine (0.06, 0.12, or 0.24 mg/kg). The strains did not differ in PR responding across the doses tested. The ACI rats demonstrated the highest behavioral output during extinction training, conditioned- and methamphetamine-primed reinstatement of previously extinguished behavior compared to the other strains. These data suggest that genetic background differentially influences extinction, conditioned reinstatement and methamphetamine-primed reinstatement in rats.

  15. Multiplexed CRISPR/Cas9- and TAR-Mediated Promoter Engineering of Natural Product Biosynthetic Gene Clusters in Yeast.

    PubMed

    Kang, Hahk-Soo; Charlop-Powers, Zachary; Brady, Sean F

    2016-09-16

    The use of DNA sequencing to guide the discovery of natural products has emerged as a new paradigm for revealing chemistries encoded in bacterial genomes. A major obstacle to implementing this approach to natural product discovery is the transcriptional silence of biosynthetic gene clusters under laboratory growth conditions. Here we describe an improved yeast-based promoter engineering platform (mCRISTAR) that combines CRISPR/Cas9 and TAR to enable single-marker multiplexed promoter engineering of large gene clusters. mCRISTAR highlights the first application of the CRISPR/Cas9 system to multiplexed promoter engineering of natural product biosynthetic gene clusters. In this method, CRISPR/Cas9 is used to induce DNA double-strand breaks in promoter regions of biosynthetic gene clusters, and the resulting operon fragments are reassembled by TAR using synthetic gene-cluster-specific promoter cassettes. mCRISTAR uses a CRISPR array to simplify the construction of a CRISPR plasmid for multiplex CRISPR and a single auxotrophic selection to improve the inefficiency of using a CRISPR array for multiplex gene cluster refactoring. mCRISTAR is a simple and generic method for multiplexed replacement of promoters in biosynthetic gene clusters that will facilitate the discovery of natural products from the rapidly growing collection of gene clusters found in microbial genome and metagenome sequencing projects.

  16. Nonsteroidal anti-inflammatory drug activated gene-1 (NAG-1) modulators from natural products as anti-cancer agents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural products are rich source of gene modulators for prevention and treatment of cancer. In recent days, nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) has been focused as a new target of diverse cancers like colorectal, pancreatic, prostate, and breast. A variety of natural...

  17. Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products

    PubMed Central

    Pelisser, Marcia Regina; Klein, Cátia Silene; Ascoli, Kelen Regina; Zotti, Thaís Regina; Arisi, Ana Carolina Maisonnave

    2009-01-01

    Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see. PMID:24031334

  18. Seasonal cardenolide production and Dop5betar gene expression in natural populations of Digitalis obscura.

    PubMed

    Roca-Pérez, Luis; Boluda, Rafael; Gavidia, Isabel; Pérez-Bermúdez, Pedro

    2004-07-01

    Productivity variations and seasonal fluctuations of cardenolides have been studied in 10 natural populations of Digitalis obscura distributed in three bioclimatic belts. Main cardenolides in D. obscura plants are those of the series A and such predominance (ca. 80-85%) over the series B metabolites is independent of the population studied or the degree of maturity of the leaves. Primary glycosides represent ca. 50-60% of total cardenolides; this percentage did not vary among populations or with the leaf age but increased in summer and decreased in winter. A correlation analysis between plant biomass and cardenolide content showed a positive relationship of these parameters, which, according to the bioclimatic distribution of the populations, suggests that certain environmental conditions may cause marked decreases in plant biomass together with a reduction in productivity. Cardenolide contents changed in the timecourse of the four seasons as a multiple response to distinct plant and/or environmental factors. The lowest production was recorded in May, followed by a fast cardenolide accumulation in summer, a decreasing phase in autumn, and a stationary phase in winter. We also analysed the seasonal expression of the gene encoding the progesterone 5beta-reductase, enzyme producing the required 5beta-configured intermediaries of cardenolides. A fragment of the isolated partial genomic sequence was used as a probe for Northern analysis to study the seasonal gene expression in selected populations. The expression pattern showed increasing levels from February to July and a further reduction in autumn, although harmful climatic conditions seems to induce overexpression of this gene.

  19. Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

    PubMed

    Li, Nan; Wang, Yuanlong; Zhu, Ping; Liu, Zhenmin; Guo, Benheng; Ren, Jing

    2015-02-01

    Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H₂O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P₂₃. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production.

  20. Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

    SciTech Connect

    VanEtten, H.

    1997-06-01

    The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes.

  1. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  2. Bayesian Computational Approaches for Gene Regulation Studies of Bioethanol and Biohydrogen Production. Final Scientific/Technical Report

    SciTech Connect

    Newberg, Lee; McCue, Lee Anne; Van Roey, Patrick

    2014-04-17

    The project developed mathematical models and first-version software tools for the understanding of gene regulation across multiple related species. The project lays the foundation for understanding how certain alpha-proteobacterial species control their own genes for bioethanol and biohydrogen production, and sets the stage for exploiting bacteria for the production of fuels. Enabling such alternative sources of fuel is a high priority for the Department of Energy and the public.

  3. Self-association of the WT1 tumor suppressor gene product

    SciTech Connect

    Bruening, W.; Nakagama, H.: Bardessy, N.

    1994-09-01

    Wilms` tumor (WT), an embryonal malignancy of the kidney, occurs most frequently in children under the age of 5 years, affecting {approximately}1 in 10,000 individuals. The WT1 tumor suppressor gene, residing at 11p13, is structurally altered in {approximately}10-15% of WT cases. Individuals with germline mutations within the WT1 gene suffer from predisposition to WT and developmental defects of the urogenital system. Patients with heterozygous deletions of the WT1 gene, or mutations predicted to cause inactivation of one WT1 allele, suffer relatively mild genital system defects (notably hypospadias and cryptorchidism in males) and a predisposition to WT. These results suggest that developing genital system development is sensitive to the absolute concentrations of the WT1 gene products. Patients with missense mutations within the WT1 gene, however, can suffer from a much more severe disorder known as Denys-Drash syndrome (DDS). This syndrome is characterized by intersex disorders, renal nephropathy, and a predisposition to WTs. The increased severity of the developmental defects associated with DDS, compared to those individuals with mild genital system anomalies and WTs, suggests that mutations defined in patients with DDS behave in a dominant-negative fashion. We have identified a novel WT1 mutation in a patient with DDS. This mutation, predicted to produce a truncated WT1 polypeptide encompassing exons 1, 2, and 3, defines a domain capable of behaving as an antimorph. We have also demonstrated that WT1 can self-associate in vivo using yeast two-hybrid systems. Deletion analysis have mapped the interacting domains to the amino terminus of the WT1 polypeptide, within exons 1 and 2. These results provide a molecular mechanism to explain how WT1 mutations can function in a dominant-negative fashion to eliminate wild-type WT1 activity, leading to DDS.

  4. Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.

    PubMed

    Li, Jin; Stoddard, Thomas J; Demorest, Zachary L; Lavoie, Pierre-Olivier; Luo, Song; Clasen, Benjamin M; Cedrone, Frederic; Ray, Erin E; Coffman, Andrew P; Daulhac, Aurelie; Yabandith, Ann; Retterath, Adam J; Mathis, Luc; Voytas, Daniel F; D'Aoust, Marc-André; Zhang, Feng

    2016-02-01

    Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

  5. Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives.

    PubMed

    Sala, Francesco; Manuela Rigano, M; Barbante, Alessandra; Basso, Barbara; Walmsley, Amanda M; Castiglione, Stefano

    2003-01-30

    Stable integration of a gene into the plant nuclear or chloroplast genome can transform higher plants (e.g. tobacco, potato, tomato, banana) into bioreactors for the production of subunit vaccines for oral or parental administration. This can also be achieved by using recombinant plant viruses as transient expression vectors in infected plants. The use of plant-derived vaccines may overcome some of the major problems encountered with traditional vaccination against infectious diseases, autoimmune diseases and tumours. They also offer a convenient tool against the threat of bio-terrorism. State of the art, experimental strategies, safety and perspectives are discussed in this article.

  6. Computational identification of gene over-expression targets for metabolic engineering of taxadiene production.

    PubMed

    Boghigian, Brett A; Armando, John; Salas, Daniel; Pfeifer, Blaine A

    2012-03-01

    Taxadiene is the first dedicated intermediate in the biosynthetic pathway of the anticancer compound Taxol. Recent studies have taken advantage of heterologous hosts to produce taxadiene and other isoprenoid compounds, and such ventures now offer research opportunities that take advantage of the engineering tools associated with the surrogate host. In this study, metabolic engineering was applied in the context of over-expression targets predicted to improve taxadiene production. Identified targets included genes both within and outside of the isoprenoid precursor pathway. These targets were then tested for experimental over-expression in a heterologous Escherichia coli host designed to support isoprenoid biosynthesis. Results confirmed the computationally predicted improvements and indicated a synergy between targets within the expected isoprenoid precursor pathway and those outside this pathway. The presented algorithm is broadly applicable to other host systems and/or product choices.

  7. Natural and Engineered Hydroxyectoine Production Based on the Pseudomonas stutzeri ectABCD-ask Gene Cluster▿ †

    PubMed Central

    Seip, Britta; Galinski, Erwin A.; Kurz, Matthias

    2011-01-01

    We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190T and evaluate the suitability of P. stutzeri DSM5190T for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in E. coli under the conditions employed exceeded that of the natural producer Pseudomonas stutzeri and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes. PMID:21169432

  8. Expression of Clostridium acetobutylicum ATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

    PubMed Central

    Bermejo, Lourdes L.; Welker, Neil E.; Papoutsakis, Eleftherios T.

    1998-01-01

    A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided. PMID:9501448

  9. Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

    PubMed Central

    2011-01-01

    Background Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. Results The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively. Conclusions Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial

  10. Abundance and distribution of Macrolide-Lincosamide-Streptogramin resistance genes in an anaerobic-aerobic system treating spiramycin production wastewater.

    PubMed

    Liu, Miaomiao; Ding, Ran; Zhang, Yu; Gao, Yingxin; Tian, Zhe; Zhang, Tong; Yang, Min

    2014-10-15

    The behaviors of the Macrolide-Lincosamide-Streptogramin (MLS) resistance genes were investigated in an anaerobic-aerobic pilot-scale system treating spiramycin (SPM) production wastewater. After screening fifteen typical MLS resistance genes with different mechanisms using conventional PCR, eight detected genes were determined by quantitative PCR, together with three mobile elements. Aerobic sludge in the pilot system exhibited a total relative abundance of MLS resistance genes (per 16S rRNA gene) 2.5 logs higher than those in control samples collected from sewage and inosine wastewater treatment systems (P < 0.05), implying the presence of SPM could induce the production of MLS resistance genes. However, the total relative gene abundance in anaerobic sludge (4.3 × 10(-1)) was lower than that in aerobic sludge (3.7 × 10(0)) despite of the higher SPM level in anaerobic reactor, showing the advantage of anaerobic treatment in reducing the production of MLS resistance genes. The rRNA methylase genes (erm(B), erm(F), erm(X)) were the most abundant in the aerobic sludge (5.3 × 10(-1)-1.7 × 10(0)), followed by esterase gene ere(A) (1.3 × 10(-1)) and phosphorylase gene mph(B) (5.7 × 10(-2)). In anaerobic sludge, erm(B), erm(F), ere(A), and msr(D) were the major ones (1.2 × 10(-2)-3.2 × 10(-1)). These MLS resistance genes (except for msr(D)) were positively correlated with Class 1 integron (r(2) = 0.74-0.93, P < 0.05), implying the significance of horizontal transfer in their proliferation.

  11. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum.

    PubMed

    Li, Huan-Jun; He, Yi-Long; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Li, Na; Xu, Jun-Wei

    2016-06-10

    The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8μg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs.

  12. Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells

    SciTech Connect

    Correll, P.H.; Fink, J.K.; Brady, R.O.; Perry, L.K.; Karlsson, S. )

    1989-11-01

    The human glucocerebrosidase (GC) gene has been transferred efficiently into spleen colony-forming unit (CFU-S) multipotential hematopoietic progenitor cells, and production of human GC RNA and protein has been achieved in transduced CFU-S colonies. High-titer retroviral vectors containing the human GC cDNA were constructed. Four vectors were compared with respect to gene-transfer efficiency into CFU-S progenitors. One vector (G vector) required high concentrations of interleukins 3 and 6 during stimulation and coculture for efficient transduction of CFU-S progenitors. The remaining three vectors (NTG, GTN, and GI vectors) transduced these progenitors at infection frequencies approaching 100% using low concentrations of hematopoietic growth factors to simulate cell division prior to and during the infection. Vectors using the viral long terminal repeat enhancer/promoter to drive the human GC cDNA produced high levels of human GC RNA in the progeny of CFU-S progenitors after gene transfer. All three vectors producing human GC RNA in CFU-S colonies can generate human GC as detected by immunochemical analysis of CFU-S colonies. The capacity of the viral long terminal repeat and the internal thymidine kinase promoter to direct synthesis of RNA in transduced bone marrow and spleen cells 5 months after bone marrow transplantation reflected the performance of these promoters in NTG-transduced CFU-S colonies.

  13. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

    PubMed

    Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; Buttini, Manuel; Linster, Carole L; Medina, Eva; Balling, Rudi; Hiller, Karsten

    2013-05-07

    Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

  14. Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product.

    PubMed

    Singh, Mangal; Chaudhary, Sandeep; Sareen, Dipti

    2017-03-01

    Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.

  15. A genetic strategy to measure circulating Drosophila insulin reveals genes regulating insulin production and secretion.

    PubMed

    Park, Sangbin; Alfa, Ronald W; Topper, Sydni M; Kim, Grace E S; Kockel, Lutz; Kim, Seung K

    2014-08-01

    Insulin is a major regulator of metabolism in metazoans, including the fruit fly Drosophila melanogaster. Genome-wide association studies (GWAS) suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM). To identify molecular functions of genes linked to T2DM risk, we developed a genetic tool to measure insulin-like peptide 2 (Ilp2) levels in Drosophila, a model organism with superb experimental genetics. Our system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the Drosophila orthologues of human T2DM risk genes, including GLIS3 and BCL11A, revealed roles of these Drosophila genes in Ilp2 production or secretion. Discovery of Drosophila mechanisms and regulators controlling in vivo insulin dynamics should accelerate functional dissection of diabetes genetics.

  16. c-Myb influences HIV type 1 gene expression and virus production.

    PubMed

    Churchill, M J; Ramsay, R G; Rhodes, D I; Deacon, N J

    2001-11-01

    c-Myb is expressed in proliferating T cells. Fifteen c-Myb-binding sites can be identified in the HIV-1 long terminal repeat (LTR), suggesting that c-Myb may regulate HIV-1 gene expression and virus replication. Increasing the cellular levels of c-Myb by transient transfection of CEM cells resulted in a 10- to 20-fold activation of HIV-1 LTR-driven gene expression and mutation of one high-affinity Myb-binding site within the LTR reduced this activation by 60 to 70%. Conversely, inhibition of c-Myb expression in MT-2 cells by treatment with c-myb antisense oligonucleotides decreased HIV-1 replication by 85%, as measured by reverse transcriptase activity and cytopathic effects. The effect of c-myb antisense oligonucleotides on HIV-1 gene expression and virus particle production appeared to be independent of cell proliferation, but dependent on the presence of c-Myb activity mediated through the HIV-1 LTR. These data show that c-myb expression affects HIV-1 replication in CD4(+) T cells.

  17. WNP: A Novel Algorithm for Gene Products Annotation from Weighted Functional Networks

    PubMed Central

    Magi, Alberto; Tattini, Lorenzo; Benelli, Matteo; Giusti, Betti; Abbate, Rosanna; Ruffo, Stefano

    2012-01-01

    Predicting the biological function of all the genes of an organism is one of the fundamental goals of computational system biology. In the last decade, high-throughput experimental methods for studying the functional interactions between gene products (GPs) have been combined with computational approaches based on Bayesian networks for data integration. The result of these computational approaches is an interaction network with weighted links representing connectivity likelihood between two functionally related GPs. The weighted network generated by these computational approaches can be used to predict annotations for functionally uncharacterized GPs. Here we introduce Weighted Network Predictor (WNP), a novel algorithm for function prediction of biologically uncharacterized GPs. Tests conducted on simulated data show that WNP outperforms other 5 state-of-the-art methods in terms of both specificity and sensitivity and that it is able to better exploit and propagate the functional and topological information of the network. We apply our method to Saccharomyces cerevisiae yeast and Arabidopsis thaliana networks and we predict Gene Ontology function for about 500 and 10000 uncharacterized GPs respectively. PMID:22761703

  18. Nucleotide and protein sequences for dog masticatory tropomyosin identify a novel Tpm4 gene product

    PubMed Central

    Reiser, Peter J.

    2016-01-01

    Jaw-closing muscles of several vertebrate species, including members of Carnivora, express a unique, “masticatory”, isoform of myosin heavy chain, along with isoforms of other myofibrillar proteins that are not expressed in most other muscles. It is generally believed that the complement of myofibrillar isoforms in these muscles serves high force generation for capturing live prey, breaking down tough plant material and defensive biting. A unique isoform of tropomyosin (Tpm) was reported to be expressed in cat jaw-closing muscle, based upon two-dimensional gel mobility, peptide mapping, and immunohistochemistry. The objective of this study was to obtain protein and gene sequence information for this unique Tpm isoform. Samples of masseter (also a jaw-closing muscle), tibialis (with predominantly fast-twitch fibers), and the deep lateral gastrocnemius (predominantly slow-twitch fibers) were obtained from adult dogs. Expressed Tpm isoforms were cloned and sequencing yielded cDNAs that were identical to genomic predicted striated muscle Tpm1.1St(a,b,b,a) (historically referred to as αTpm), Tpm2.2St(a,b,b,a) (βTpm) and Tpm3.12St(a,b,b,a) (cTpm) isoforms (nomenclature reflects predominant tissue expression (“St”—striated muscle) and exon splicing pattern), as well as a novel 284 amino acid isoform observed in jaw-closing muscle that is identical to a genomic predicted product of the Tpm4 gene (δTpm) family. The novel isoform is designated as Tpm4.3St(a,b,b,a). The myofibrillar Tpm isoform expressed in dog masseter exhibits a unique electrophoretic mobility on gels containing 6 M urea, compared to other skeletal Tpm isoforms. To validate that the cloned Tpm4.3 isoform is the Tpm expressed in dog masseter, E. coli-expressed Tpm4.3 was electrophoresed in the presence of urea. Results demonstrate that Tpm4.3 has identical electrophoretic mobility to the unique dog masseter Tpm isoform and is of different mobility from that of muscle Tpm1.1, Tpm2.2 and Tpm3

  19. Essential Oils Modulate Gene Expression and Ochratoxin A Production in Aspergillus carbonarius

    PubMed Central

    El Khoury, Rachelle; Atoui, Ali; Verheecke, Carol; Maroun, Richard; El Khoury, Andre; Mathieu, Florence

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin, mainly produced on grapes by Aspergillus carbonarius, that causes massive health problems for humans. This study aims to reduce the occurrence of OTA by using the ten following essential oils (E.Os): fennel, cardamom, anise, chamomile, celery, cinnamon, thyme, taramira, oregano and rosemary at 1 µL/mL and 5 µL/mL for each E.O.As a matter of fact, their effects on the OTA production and the growth of A. carbonarius S402 cultures were evaluated, after four days at 28 °C on a Synthetic Grape Medium (SGM). Results showed that A. carbonarius growth was reduced up to 100%, when cultured with the E.Os of cinnamon, taramira, and oregano at both concentrations and the thyme at 5 µL/mL. As for the other six E.Os, their effect on A. carbonarius growth was insignificant, but highly important on the OTA production. Interestingly, the fennel E.O at 5 µL/mL reduced the OTA production up to 88.9% compared to the control, with only 13.8% of fungal growth reduction. We further investigated the effect of these E.Os on the expression levels of the genes responsible for the OTA biosynthesis (acOTApks and acOTAnrps along with the acpks gene) as well as the two regulatory genes laeA and vea, using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) method. The results revealed that these six E.Os reduced the expression of the five studied genes, where the ackps was downregulated by 99.2% (the highest downregulation in this study) with 5 µL/mL of fennel E.O.As for the acOTApks, acOTAnrps, veA and laeA, their reduction levels ranged between 10% and 96% depending on the nature of the E.O and its concentration in the medium. PMID:27548221

  20. Enhancement of doxorubicin production by expression of structural sugar biosynthesis and glycosyltransferase genes in Streptomyces peucetius.

    PubMed

    Malla, Sailesh; Niraula, Narayan Prasad; Liou, Kwangkyoung; Sohng, Jae Kyung

    2009-08-01

    To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes desIII and desIV from Streptomyces venezuelae ATCC 15439 and the glycosyltransferase pair dnrS/dnrQ from Streptomyces peucetius ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong ermE promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of dnrS and the dnrS/dnrQ pair, whereas pDesSD25 and pDesQS25 were constructed to express desIII/desIV and dnrS/dnrQ-desIII/desIV, respectively. All of these recombinant plasmids were introduced into S. peucetius ATCC 27952. The recombinant strains produced more DXR than the S. peucetius parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.

  1. Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu

    SciTech Connect

    Kondou, Youhei; Kitazawa, Daisuke; Takeda, Shigeki; Yamashita, Eiki; Mizuguchi, Mineyuki; Kawano, Keiichi; Tsukihara, Tomitake

    2005-01-01

    Bacteriophage Mu baseplate protein gene product 44 was crystallized. The crystal belongs to space group R3, with unit-cell parameters a = b = 126.6, c = 64.2 Å. Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-rays to at least 2.1 Å resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.

  2. Tissue Engineering for Bone Production- Stem Cells, Gene Therapy and Scaffolds

    PubMed Central

    Khaled, E.G; Saleh, M; Hindocha, S; Griffin, M; Khan, Wasim S

    2011-01-01

    A bone graft has been the gold standard treatment for repairing bone defects. However, due to bone grafts associated donor site morbidity several alternative bone substitutes options have been made available but with their added expense and limited osteoinductive properties they are not ideal. Therefore, research has begun in tissue engineering to investigate stem cells, which are one of the body’s own mechanisms used to repair bone. Stem cells are clonogenic undifferentiated cells capable of self-renewal. Readily available from numerous of sources stem cells have the potential to differentiate in osteoblasts and chrondrocytes showing capability to repair both bone and cartilage. The known immunologic properties of stem cells further enhance their therapeutic appeal. Stem cells have shown to be excellent carriers for gene transfer having the capability to be transduced. Gene transfer could enable growth factors and bone morphogentic proteins to enhance bone repair. Stem cells are implanted onto scaffolds, which are structures capable of supporting tissue formation by allowing cell migration, proliferation and differentiation. Research aims to produce scaffolds that deliver and retain cells, allow for cell attachment has adequate biodegradability, biocompatibility and non-immunogenicity. However, having tried and testing numerous materials including synthetic and natural products research into the perfect scaffold product continues. This review aims to explain how stem cells were discovered, the techniques used to isolate stem cells, identify and manipulate them down different cell lineages and discuss the research into using stem cells to reconstruct bone using genetic modification and scaffolds. PMID:21886695

  3. Effect of retS gene on antibiotics production in Pseudomonas fluorescens FD6.

    PubMed

    Zhang, Qingxia; Xiao, Qi; Xu, Jingyou; Tong, Yunhui; Wen, Jia; Chen, Xijun; Wei, Lihui

    2015-11-01

    A hybrid sensor kinase termed RetS (regulator of exopolysaccharide and Type III secretion) controls expression of numerous genes in Pseudomonas aeruginosa. To investigate the function of RetS in P. fluorescens FD6, the retS gene was disrupted. Genetic inactivation of retS resulted in enhanced production of 2, 4-diacetylphloroglucinol, pyrrolnitrin, and pyoluteorin. The retS mutant also exhibited significant increase in phlA-lacZ, prnA-lacZ, and pltA-lacZ transcription levels, influencing expression levels of the small regulatory RNAs RsmX and RsmZ. In the gacSretS double mutant, all the phenotypic changes caused by the retS deletion were reversed to the level of gacS single mutant. Furthermore, the retS mutation drastically elevated biofilm formation and improved the colonization ability of strain FD6 on wheat rhizospheres. Based on these results, we proposed that RetS negatively controlled the production of antibiotics through the Gac/Rsm pathway in P. fluorescens FD6.

  4. Quantification of Tri5 gene, expression, and deoxynivalenol production during the malting of barley.

    PubMed

    Vegi, Anuradha; Schwarz, Paul; Wolf-Hall, Charlene E

    2011-11-01

    Fusarium can survive, grow, and produce mycotoxins during malting. We evaluated the percentage of barley kernels infected with Fusarium (FI) and deoxynivalenol (DON) concentration in three barley treatments (high-quality, naturally infected, and Fusarium graminearum inoculated barley) during various stages of malting. We also applied real-time polymerase chain reaction (real-time PCR) and real-time reverse transcriptase PCR (real-time RT-PCR) methods to quantify trichothecene-producing (Tri5) DNA concentration and expression, respectively. We observed that FI significantly (P<0.05) increased during the germination stage of malting in all barley treatments. Temperatures of 49°C and higher during kilning reduced the FI in high-quality barley treatments, but for inoculated treatments temperatures in excess of 60°C were needed to reduce FI. The Tri5 DNA concentration ranged from non-detectable to 3.9 ng/50mg, 0.1 to 109.8 ng/50mg and 3.4 to 397.5 ng/50 mg in malted high-quality, inoculated and naturally infected barley treatments respectively. Strong gene expression (Tri5) in naturally infected barley treatments was found during the third day of germination, when compared to high-quality and inoculated barley treatments during malting. Deoxynivalenol was present even at high kilning temperatures, as DON is heat stable. The average DON concentration ranged from non-detectable to 0.1 μg/g, non-detectable to 1.1 μg/g, and 1.5 to 45.9 μg/g during various stages of malting in high-quality, inoculated and infected barley and malt samples respectively. Overall, the last 2 days of germination and initial stages of kilning were peak stages for FI, Tri5 gene production, Tri5 gene expression and DON production.

  5. Characterization and expression of the human rhoH12 gene product

    SciTech Connect

    Avraham, H.; Weinberg, R.A.

    1989-05-01

    The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in the place of the glycine and leucine in place of the gutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via contransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho proteins was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.

  6. [Improvement of natamycin production in an industrial strain by heterologous expression of the afsRS(cla) global regulatory genes].

    PubMed

    Tao, Zhengsheng; Wang, Yemin; Zheng, Hualiang; Tao, Meifeng

    2015-05-01

    The afsRS(cla) global regulatory genes from Streptomyces clavuligerus activate the production of two antibiotics in Streptomyces lividans. In this study, we gained an increase of 38% in the production of natamycin (3.56 g/L) in an industrial strain Streptomyces gilvosporeus TZ1401 through the integration of pHL851 that bears the afsRS(cla) global regulatory genes into its genome. We discovered by quantitive real-time reverse transcription PCR (qRT-PCR) that the expression of 6 genes of the natamycin biosynthetic gene cluster were improved from 1.9 to 2.7 times. This suggests that afsRS(cla) improve the production of natamycin through increased transcription. This study provides a good example for applying afsRS(cla) in high yield breeding of industrial antibiotic producers.

  7. Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae.

    PubMed Central

    Reynolds, A E; Murray, A W; Szostak, J W

    1987-01-01

    We have examined the replication and segregation of the Saccharomyces cerevisiae 2 microns circle. The amplification of the plasmid at low copy numbers requires site-specific recombination between the 2 microns inverted repeat sequences catalyzed by the plasmid-encoded FLP gene. No other 2 microns gene products are required. The overexpression of FLP in a strain carrying endogenous 2 microns leads to uncontrolled plasmid replication, longer cell cycles, and cell death. Two different assays show that the level of Flp activity decreases with increasing 2 microns copy number. This regulation requires the products of the REP1 and REP2 genes. These gene products also act together to ensure that 2 microns molecules are randomly segregated between mother and daughter cells at cell division. Images PMID:3316982

  8. Mutational Analysis of the sbo-alb Locus of Bacillus subtilis: Identification of Genes Required for Subtilosin Production and Immunity

    PubMed Central

    Zheng, Guolu; Hehn, Robin; Zuber, Peter

    2000-01-01

    The Bacillus subtilis 168 derivative JH642 produces a bacteriocin, subtilosin, which possesses activity against Listeria monocytogenes. Inspection of the amino acid sequence of the presubtilosin polypeptide encoded by the gene sboA and sequence data from analysis of mature subtilosin indicate that the precursor subtilosin peptide undergoes several unique and unusual chemical modifications during its maturation process. The genes of the sbo-alb operon are believed to function in the synthesis and maturation of subtilosin. Nonpolar mutations introduced into each of the alb genes resulted in loss or reduction of subtilosin production. sboA, albA, and albF mutants showed no antilisterial activity, indicating that the products of these genes are critical for the production of active subtilosin. Mutations in albB, -C, and -D resulted in reduction of antilisterial activity and decreased immunity to subtilosin, particularly under anaerobic conditions. A new gene, sboX, encoding another bacteriocin-like product was discovered residing in a sequence overlapping the coding region of sboA. Construction of an sboX-lacZ translational fusion and analysis of its expression indicate that sboX is induced in stationary phase of anaerobic cultures of JH642. An in-frame deletion of the sboX coding sequence did not affect the antilisterial activity or production of or immunity to subtilosin. The results of this investigation show that the sbo-alb genes are required for the mechanisms of subtilosin synthesis and immunity. PMID:10809709

  9. Cocktail δ-integration of xylose assimilation genes for efficient ethanol production from xylose in Saccharomyces cerevisiae.

    PubMed

    Kato, Hiroko; Matsuda, Fumio; Yamada, Ryosuke; Nagata, Kento; Shirai, Tomokazu; Hasunuma, Tomohisa; Kondo, Akihiko

    2013-09-01

    Cocktail δ-integration was applied to improve ethanol production from xylose in Saccharomyces cerevisiae. Two hundred of recombinant S. cerevisiae strains possessing various copies of XYL1, XYL2, and XKS1 genes were constructed by cocktail δ-integration. Efficient strains with efficient ethanol production from xylose were successfully obtained by the fermentation test.

  10. Diversity, Distribution and Quantification of Antibiotic Resistance Genes in Goat and Lamb Slaughterhouse Surfaces and Meat Products

    PubMed Central

    Lavilla Lerma, Leyre; Benomar, Nabil; Knapp, Charles W.; Correa Galeote, David; Gálvez, Antonio; Abriouel, Hikmate

    2014-01-01

    The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated ‘hot spots.’ The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination. PMID:25479100

  11. From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028

    PubMed Central

    Greule, Anja; Zhang, Songya; Paululat, Thomas; Bechthold, Andreas

    2017-01-01

    Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods. Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound. The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes Tü6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism. PMID:28117820

  12. From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028.

    PubMed

    Greule, Anja; Zhang, Songya; Paululat, Thomas; Bechthold, Andreas

    2017-01-13

    Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods. Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound. The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes Tü6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

  13. Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

    PubMed Central

    Chen, Jianming

    2015-01-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  14. Immunological characterization of the gag gene products of bovine immunodeficiency virus.

    PubMed Central

    Battles, J K; Hu, M Y; Rasmussen, L; Tobin, G J; Gonda, M A

    1992-01-01

    The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed. Images PMID:1331499

  15. Immunological characterization of the gag gene products of bovine immunodeficiency virus.

    PubMed

    Battles, J K; Hu, M Y; Rasmussen, L; Tobin, G J; Gonda, M A

    1992-12-01

    The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed.

  16. A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris

    PubMed Central

    Protasevich, Irina I.; Brouillette, Christie G.; Harrell, Patina M.; Hildebrandt, Ellen; Gasser, Brigitte; Mattanovich, Diethard; Ward, Andrew; Chang, Geoffrey; Urbatsch, Ina L.

    2011-01-01

    Background Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers. Methodology/Principal Findings Codon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from Tm ∼40°C to 49°C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids. Conclusion The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins. PMID:21826197

  17. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

  18. Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus aureus strains harboring seh gene.

    PubMed

    Schubert, Justyna; Podkowik, Magdalena; Bystroń, Jarosław; Bania, Jacek

    2016-10-17

    Twenty Staphylococcus aureus strains harboring seh gene, including one carrying also sec gene and 11 sea gene, were grown in BHI+YE broth and milk and were tested for SEA, SEC and SEH production. All strains decreased pH of BHI+YE broth at 24h and increased them at 48h. Seventeen S. aureus strains grown in milk changed pH for no >0.3 unit until 48h. Three other S. aureus strains significantly decreased pH during growth in milk. All S. aureus produced SEH in BHI+YE broth in amounts ranging from 95 to 1292ng/ml, and from 170 to 4158ng/ml at 24 and 48h, respectively. SEH production in milk by 17 strains did not exceed 23ng/ml at 24h and 36ng/ml at 48h. Three S. aureus strains able to decrease milk pH produced 107-3029ng/ml and 320-4246ng/ml of SEH in milk at 24 and 48h, respectively. These strains were grown in milk and BHI+YE broth with pH stabilized at values near neutral leading to a significant decrease of SEH production. Representative weak SEH producers were grown in milk at reduced pH resulting in moderate increase in SEH production. SEA was produced in milk by 10S. aureus strains at 24-151ng/ml at 24h, and 31-303ng/ml at 48h. SEA production in milk was higher or comparable as in BHI+YE broth in 3 strains and lower for remaining strains. Production of SEC by sec-positive S. aureus strains was lower in milk than in BHI+YE broth, ranging from 131 to 2319ng/ml at 24 and 48h in milk and 296-30,087ng/ml in BHI+YE at 24 and 48h. Both lacE and lacG transcripts involved in lactose metabolism were significantly up-regulated in milk in strong SEH producers. In these strains hld, rot and sarA transcripts were up-regulated in milk as compared to weak SEH producers. Stabilization of milk pH at a value of raw milk significantly down-regulated hld, rot and sarA RNA in strong SEH producers. Milk was generally found unfavorable for enterotoxin production. However, certain S. aureus strains were not restricted in SEH and SEA expression in milk, unlike SEC which remained down

  19. Increasing Avermectin Production in Streptomyces avermitilis by Manipulating the Expression of a Novel TetR-Family Regulator and Its Target Gene Product

    PubMed Central

    Liu, Wenshuai; Zhang, Qinling; Guo, Jia; Chen, Zhi; Li, Jilun

    2015-01-01

    Avermectins produced by Streptomyces avermitilis are commercially important anthelmintic agents. The detailed regulatory mechanisms of avermectin biosynthesis remain unclear. Here, we identified SAV3619, a TetR-family transcriptional regulator designated AveT, to be an activator for both avermectin production and morphological differentiation in S. avermitilis. AveT was shown to indirectly stimulate avermectin production by affecting transcription of the cluster-situated activator gene aveR. AveT directly repressed transcription of its own gene (aveT), adjacent gene pepD2 (sav_3620), sav_7490 (designated aveM), and sav_7491 by binding to an 18-bp perfect palindromic sequence (CGAAACGKTKYCGTTTCG, where K is T or G and Y is T or C and where the underlining indicates inverted repeats) within their promoter regions. aveM (which encodes a putative transmembrane efflux protein belonging to the major facilitator superfamily [MFS]), the important target gene of AveT, had a striking negative effect on avermectin production and morphological differentiation. Overexpression of aveT and deletion of aveM in wild-type and industrial strains of S. avermitilis led to clear increases in the levels of avermectin production. In vitro gel-shift assays suggested that C-5–O-B1, the late pathway precursor of avermectin B1, acts as an AveT ligand. Taken together, our findings indicate positive-feedback regulation of aveT expression and avermectin production by a late pathway intermediate and provide the basis for an efficient strategy to increase avermectin production in S. avermitilis by manipulation of AveT and its target gene product, AveM. PMID:26002902

  20. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    PubMed Central

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  1. deadpan, an essential pan-neural gene in Drosophila, encodes a helix-loop-helix protein similar to the hairy gene product.

    PubMed

    Bier, E; Vaessin, H; Younger-Shepherd, S; Jan, L Y; Jan, Y N

    1992-11-01

    Neural precursor cells in Drosophila acquire their identity early during their formation. In an attempt to determine whether all neural precursors share a set of genetic machinery, perhaps to control properties of differentiation common to all neurons, we used the enhancer-trap method to identify several genes (pan-neural genes) that are expressed in all neurons and/or their precursors. One of the pan-neural genes is deadpan, which encodes a helix-loop-helix protein closely related to the product of the segmentation gene hairy. The function of deadpan is essential for viability and is likely to be involved in the functional rather than the morphological differentiation of neurons.

  2. High-affinity L-arabinose transport operon. Gene product expression and mRNAs.

    PubMed

    Horazdovsky, B F; Hogg, R W

    1987-09-05

    Various portions of the "high-affinity" L-arabinose transport operon were cloned into the plasmid expression vector pKK223-3 and the operon-encoded protein products were identified. The results indicate that three proteins are encoded by this operon. The first is a 33,000 Mr protein that is the product of the promoter-proximal L-arabinose binding protein coding sequence, araF. A 52,000 Mr protein is encoded by sequence 3' to araF and has been assigned to the araG locus. The sequence 3' to araG encodes a 31,000 Mr protein that has been assigned to the araH locus. Both the araG and araH gene products are localized in the membrane fraction of the cell, implying a role in the membrane-associated complex of the high-affinity L-arabinose transport system. Nuclease S1 protection studies indicate that two operon message populations are present in the cell, a full-length operon transcript and a seven- to tenfold more abundant binding protein-specific message. The relative abundance of these two message populations correlates with the differential expression of the binding protein and the membrane-associated proteins of the transport system.

  3. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis

    PubMed Central

    Piriya, P. Sobana; Vasan, P. Thirumalai; Padma, V. S.; Vidhyadevi, U.; Archana, K.; Vennison, S. John

    2012-01-01

    The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production. PMID:22919503

  4. Nitric oxide production and NO synthase gene expression contribute to vascular regulation during exercise.

    PubMed

    Shen, W; Zhang, X; Zhao, G; Wolin, M S; Sessa, W; Hintze, T H

    1995-08-01

    Nitric oxide (NO) is a vasodilator produced under normal physiologic conditions primarily by the vascular endothelium lining all blood vessels. The primary stimulus for the production of nitric oxide by the constitutive endothelial nitric oxide synthase (ECNOS, Type II) found in blood vessels is most likely the shear stress, the frictional force, caused by blood flowing through blood vessels. During exercise there is an increase in cardiac output and redistribution of blood flow to increase blood flow in skeletal muscle and in the coronary circulation. These adjustments provide increased oxygen delivery to support aerobic energy production and to sustain the exercise response. NO may be involved in the regulation of vascular tone in exercising skeletal and cardiac muscle by promoting, enhancing the metabolic vasodilation. In addition, the production of NO by capillary endothelium may regulate oxygen consumption by mitochondria through chemical interactions between NO and the iron-sulfur center of these enzymes. Finally, brief exercise training may alter the gene expression for the enzyme, the constitutive endothelial NO synthase, which forms NO and may be part of the vascular adaptation seen after aerobic exercise training. Furthermore, if there is a genetic predisposition to produce NO, as in world class athletes or animals bred to race, NO may contribute to spectacular exercise performance. These three potential roles of NO will be discussed and data presented to support each of these in our review.

  5. Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9

    PubMed Central

    Nielsen, Maria Lund; Isbrandt, Thomas; Rasmussen, Kasper Bøwig; Thrane, Ulf; Hoof, Jakob Blæsbjerg; Larsen, Thomas Ostenfeld; Mortensen, Uffe Hasbro

    2017-01-01

    The full potential of fungal secondary metabolism has until recently been impeded by the lack of universal genetic tools for most species. However, the emergence of several CRISPR-Cas9-based genome editing systems adapted for several genera of filamentous fungi have now opened the doors for future efforts in discovery of novel natural products and elucidation and engineering of their biosynthetic pathways in fungi where no genetic tools are in place. So far, most studies have focused on demonstrating the performance of CRISPR-Cas9 in various fungal model species, and recently we presented a versatile CRISPR-Cas9 system that can be successfully applied in several diverse Aspergillus species. Here we take it one step further and show that our system can be used also in a phylogenetically distinct and largely unexplored species from the genus of Talaromyces. Specifically, we exploit CRISPR-Cas9-based genome editing to identify a new gene in T. atroroseus responsible for production of polyketide-nonribosomal peptide hybrid products, hence, linking fungal secondary metabolites to their genetic origin in a species where no genetic engineering has previously been performed. PMID:28056079

  6. The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis

    PubMed Central

    Liu, Jing; Hua, Wei; Yang, Hong-Li; Zhan, Gao-Miao; Deng, Lin-Bin; Wang, Xin-Fa; Liu, Gui-Hua; Wang, Han-Zhong

    2012-01-01

    Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level. PMID:22442419

  7. The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis.

    PubMed

    Liu, Jing; Hua, Wei; Yang, Hong-Li; Zhan, Gao-Miao; Li, Rong-Jun; Deng, Lin-Bin; Wang, Xin-Fa; Liu, Gui-Hua; Wang, Han-Zhong

    2012-06-01

    Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.

  8. Trade-off between constitutive and inducible resistance against herbivores is only partially explained by gene expression and glucosinolate production.

    PubMed

    Rasmann, Sergio; Chassin, Estelle; Bilat, Julia; Glauser, Gaétan; Reymond, Philippe

    2015-05-01

    The hypothesis that constitutive and inducible plant resistance against herbivores should trade-off because they use the same resources and impose costs to plant fitness has been postulated for a long time. Negative correlations between modes of deployment of resistance and defences have been observed across and within species in common garden experiments. It was therefore tested whether that pattern of resistance across genotypes follows a similar variation in patterns of gene expression and chemical defence production. Using the genetically tractable model Arabidopsis thaliana and different modes of induction, including the generalist herbivore Spodoptera littoralis, the specialist herbivore Pieris brassicae, and jasmonate application, constitutive and inducibility of resistance was measured across seven A. thaliana accessions that were previously selected based on constitutive levels of defence gene expression. According to theory, it was found that modes of resistance traded-off among accessions, particularly against S. littoralis, in which accessions investing in high constitutive resistance did not increase it substantially after attack and vice-versa. Accordingly, the average expression of eight genes involved in glucosinolate production negatively predicted larval growth across the seven accessions. Glucosinolate production and genes related to defence induction on healthy and herbivore-damaged plants were measured next. Surprisingly, only a partial correlation between glucosinolate production, gene expression, and the herbivore resistance results was found. These results suggest that the defence outcome of plants against herbivores goes beyond individual molecules or genes but stands on a complex network of interactions.

  9. Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

    PubMed Central

    Zivkovic, Milica; Miljkovic, Marija; Ruas-Madiedo, Patricia; Strahinic, Ivana; Tolinacki, Maja; Golic, Natasa

    2014-01-01

    Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11. PMID:25527533

  10. Peroxisome proliferator-activated receptor γ controls ingestive behavior, agouti-related protein, and neuropeptide Y mRNA in the arcuate hypothalamus.

    PubMed

    Garretson, John T; Teubner, Brett J W; Grove, Kevin L; Vazdarjanova, Almira; Ryu, Vitaly; Bartness, Timothy J

    2015-03-18

    Peroxisome proliferator-activated receptor γ (PPARγ) is clinically targeted for type II diabetes treatment; however, rosiglitazone (ROSI), a PPARγ agonist, increases food intake and body/fat mass as side-effects. Mechanisms for these effects and the role of PPARγ in feeding are not understood. Therefore, we tested this role in Siberian hamsters, a model of human energy balance, and C57BL/6 mice. We tested the following: (1) how ROSI and/or GW9662 (2-chloro-5-nitro-N-phenylbenzamide; PPARγ antagonist) injected intraperitoneally or into the third ventricle (3V) affected Siberian hamster feeding behaviors; (2) whether food deprivation (FD) co-increases agouti-related protein (AgRP) and PPARγ mRNA expression in Siberian hamsters and mice; (3) whether intraperitoneally administered ROSI increases AgRP and NPY in ad libitum-fed animals; (4) whether intraperitoneally administered PPARγ antagonism blocks FD-induced increases in AgRP and NPY; and finally, (5) whether intraperitoneally administered PPARγ modulation affects plasma ghrelin. Third ventricular and intraperitoneally administered ROSI increased food hoarding and intake for 7 d, an effect attenuated by 3V GW9662, and also prevented (intraperitoneal) FD-induced feeding. FD hamsters and mice increased AgRP within the arcuate hypothalamic nucleus with concomitant increases in PPARγ exclusively within AgRP/NPY neurons. ROSI increased AgRP and NPY similarly to FD, and GW9662 prevented FD-induced increases in AgRP and NPY in both species. Neither ROSI nor GW9662 affected plasma ghrelin. Thus, we demonstrated that PPARγ activation is sufficient to trigger food hoarding/intake, increase AgRP/NPY, and possibly is necessary for FD-induced increases in feeding and AgRP/NPY. These findings provide initial evidence that FD-induced increases in AgRP/NPY may be a direct PPARγ-dependent process that controls ingestive behaviors.

  11. Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

    PubMed

    Nishiyama, Yoshihiro; Nakayama, Shouta M M; Watanabe, Kensuke P; Kawai, Yusuke K; Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2016-05-03

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

  12. Gene network analysis identifies rumen epithelial cell proliferation, differentiation and metabolic pathways perturbed by diet and correlated with methane production

    PubMed Central

    Xiang, Ruidong; McNally, Jody; Rowe, Suzanne; Jonker, Arjan; Pinares-Patino, Cesar S.; Oddy, V. Hutton; Vercoe, Phil E.; McEwan, John C.; Dalrymple, Brian P.

    2016-01-01

    Ruminants obtain nutrients from microbial fermentation of plant material, primarily in their rumen, a multilayered forestomach. How the different layers of the rumen wall respond to diet and influence microbial fermentation, and how these process are regulated, is not well understood. Gene expression correlation networks were constructed from full thickness rumen wall transcriptomes of 24 sheep fed two different amounts and qualities of a forage and measured for methane production. The network contained two major negatively correlated gene sub-networks predominantly representing the epithelial and muscle layers of the rumen wall. Within the epithelium sub-network gene clusters representing lipid/oxo-acid metabolism, general metabolism and proliferating and differentiating cells were identified. The expression of cell cycle and metabolic genes was positively correlated with dry matter intake, ruminal short chain fatty acid concentrations and methane production. A weak correlation between lipid/oxo-acid metabolism genes and methane yield was observed. Feed consumption level explained the majority of gene expression variation, particularly for the cell cycle genes. Many known stratified epithelium transcription factors had significantly enriched targets in the epithelial gene clusters. The expression patterns of the transcription factors and their targets in proliferating and differentiating skin is mirrored in the rumen, suggesting conservation of regulatory systems. PMID:27966600

  13. Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli.

    PubMed Central

    Gibello, A; Ferrer, E; Sanz, J; Martin, M

    1995-01-01

    The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin. PMID:8534083

  14. A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

    PubMed

    Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

    2000-02-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

  15. Relationship between brassinosteroids and genes controlling stomatal production in the Arabidopsis hypocotyl.

    PubMed

    Fuentes, Sonia; Cañamero, Roberto C; Serna, Laura

    2012-01-01

    Stomata are excellent model systems for examining the mechanisms that regulate cell fate determination and pattern formation. It has recently been demonstrated that brassinosteroids control stomatal development by regulating both the MAPK kinase kinase YODA and the basic helix-loop-helix transcriptional factor SPEECHLESS. Here, we show that these plant regulators positively regulate stomatal formation in the hypocotyl and also accelerate their development. Hormone tests, reporter gene studies and mutant analyses revealed that brassinosteroids act upstream of the transcriptional factors CAPRICE and GLABRA2. These plant regulators control an earlier stage of stomatal production than those regulated by the membrane receptor TOO MANY MOUTHS. This work highlights differences in the genetic control of stomatal development between cotyledons or leaves and hypocotyls.

  16. Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli.

    PubMed

    Nielsen, Morten T; Madsen, Karina M; Seppälä, Susanna; Christensen, Ulla; Riisberg, Lone; Harrison, Scott J; Møller, Birger Lindberg; Nørholm, Morten H H

    2015-03-20

    Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing, and collaborative efforts such as the iGEM competition. All of these attributes facilitate exploitation of the wealth of genetic information made available by genome and RNA sequencing. Standardization also comes at the cost of reduced flexibility. We addressed this paradox by formulating a set of design principles aimed at maximizing standardization while maintaining high flexibility in choice of cloning technique and minimizing the impact of standard sequences. The design principles were applied to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable and simplifies the experimental design of projects aimed at biosynthetic pathway construction or engineering.

  17. Gene Expression Profiles in a Rabbit Model of Systemic Lupus Erythematosus Autoantibody Production1

    PubMed Central

    Rai, Geeta; Ray, Satyajit; Milton, Jacqueline; Yang, Jun; Ren, Ping; Lempicki, Richard; Mage, Rose G.

    2010-01-01

    We previously reported the establishment of a rabbit (Oryctolagus cuniculus) model in which peptide immunization led to production of lupus-like autoantibodies including anti-Sm, -RNP, -SS-A, -SS-B and –dsDNA characteristic of those produced in Systemic Lupus Erythematosus (SLE) patients. Some neurological symptoms in form of seizures and nystagmus were observed. The animals used in the previous and in the present study were from a National Institute of Allergy and Infectious Diseases colony of rabbits that were pedigreed, immunoglobulin allotype-defined but not inbred. Their genetic heterogeneity may correspond to that found among patients of a given ethnicity. We extended the information about this rabbit model by microarray based expression profiling. We first demonstrated that human expression arrays could be used with rabbit RNA to yield information on molecular pathways. We then designed a study evaluating gene expression profiles in 8 groups of control and treated rabbits (47 rabbits in total). Genes significantly upregulated in treated rabbits were associated with NK cytotoxicity, antigen presentation, leukocyte migration, cytokine activity, protein kinases, RNA spliceosomal ribonucleoproteins, intracellular signaling cascades, and glutamate receptor activity. These results link increased immune activation with up-regulation of components associated with neurological and anti-RNP responses, demonstrating the utility of the rabbit model to uncover biological pathways related to SLE-induced clinical symptoms, including Neuropsychiatric Lupus. Our finding of distinct gene expression patterns in rabbits that made anti-dsDNA compared to those that only made other anti-nuclear antibodies should be further investigated in subsets of SLE patients with different autoantibody profiles. PMID:20817871

  18. Advanced Glycation End-Products affect transcription factors regulating insulin gene expression

    SciTech Connect

    Puddu, A.; Storace, D.; Odetti, P.; Viviani, G.L.

    2010-04-23

    Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic {beta}-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation prevents FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.

  19. Distribution of the Sex combs reduced Gene Products in Drosophila melanogaster

    PubMed Central

    Mahaffey, James W.; Kaufman, Thomas C.

    1987-01-01

    The spatial and temporal distribution of RNA and protein encoded by the homeotic Sex combs reduced (Scr) gene were examined during Drosophila development. The gene products are present in the epidermis of both the labial and first thoracic segments as would be predicted from prior genetic studies. However, the pattern in the central nervous system (CNS) and mesoderm is further restricted; the major expression located in the labial neuromere of the CNS and the mesoderm of the first thoracic segment. The spatial restriction within the CNS is correlated with and may be due to a differential timing of expression in the labial and first thoracic ectoderm. The labial ectoderm accumulates the Scr RNA prior to segregation of the neuroblasts while expression in the first thoracic ectoderm occurs after neuroblast segregation. The protein is also observed in the subesophageal ganglia of both larvae and adults, as well as in the labial and first thoracic imaginal discs. Surprisingly, the protein is also present to a lesser extent in second and third thoracic leg discs. PMID:3117618

  20. Genetic Characterization of the Homeodomain-Independent Activity of the Drosophila Fushi Tarazu Gene Product

    PubMed Central

    Hyduk, D.; Percival-Smith, A.

    1996-01-01

    The gene product of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is a first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results. PMID:8852847

  1. The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product.

    PubMed Central

    Refaeli, Y; Levy, D N; Weiner, D B

    1995-01-01

    The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection. Vpr protein induced nuclear translocation of Rip-1, as did glucocorticoid receptor (GR)-II-stimulating steroids. Importantly, Vpr and Rip-1 coimmunoprecipitated with the human GR as part of an activated receptor complex. Vpr complementation of a vpr mutant virus was also mimicked by GR-II-stimulating steroids. Vpr and GR-II actions were inhibited by mifepristone, a GR-II pathway inhibitor. Together these data directly link the activity of the vpr gene product to the glucocorticoid steroid pathway and provide a biochemical mechanism for the cellular and viral activity of Vpr, as well as suggest that a unique class of antivirals, which includes mifepristone (RU486), may influence HIV-1 replication. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:7724608

  2. The product of the adenovirus intermediate gene IX is a transcriptional activator.

    PubMed Central

    Lutz, P; Rosa-Calatrava, M; Kedinger, C

    1997-01-01

    We have investigated the functional properties of the product of the adenovirus type 5 gene IX. This gene, which is expressed at intermediate times postinfection, encodes a small polypeptide (pIX) of 140 residues that has previously been shown to be incorporated into the viral capsid. Here, we show that pIX, in addition to its structural contribution, exhibits transcriptional properties. In transient transfection experiments, expression of pIX stimulated adenovirus major late promoter activity. The effect was independent of other viral proteins, but the level of promoter activation appeared strongly pIX dose dependent; similar levels of induction were observed with other cellular or viral TATA-containing (but not with TATA-less) promoters. This promoter specificity could be reproduced in a cell-free transcription system by the addition of purified recombinant pIX, further stressing the transcriptional nature of the phenomenon. A preliminary structural analysis of pIX indicated that the integrity of a putative leucine zipper at the carboxy-terminal end of the molecule, as well as elements within the amino-terminal half, was critical for pIX transcriptional activity. The relevance of these findings in adenovirus infection is discussed. PMID:9188576

  3. The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

    PubMed Central

    Cangiano, Giuseppina; Sirec, Teja; Panarella, Cristina; Isticato, Rachele; Baccigalupi, Loredana; De Felice, Maurilio

    2014-01-01

    The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform. PMID:25239894

  4. Spatial and Temporal Variations in Chitinolytic Gene Expression and Bacterial Biomass Production during Chitin Degradation

    PubMed Central

    Baty, Ace M.; Eastburn, Callie C.; Techkarnjanaruk, Somkiet; Goodman, Amanda E.; Geesey, Gill G.

    2000-01-01

    Growth of the chitin-degrading marine bacterium S91 on solid surfaces under oligotrophic conditions was accompanied by the displacement of a large fraction of the surface-derived bacterial production into the flowing bulk aqueous phase, irrespective of the value of the surface as a nutrient source. Over a 200-h period of surface colonization, 97 and 75% of the bacterial biomass generated on biodegradable chitin and a nonnutritional silicon surface, respectively, detached to become part of the free-living population in the bulk aqueous phase. Specific surface-associated growth rates that included the cells that subsequently detached from the substrata varied depending on the nutritional value of the substratum and during the period of surface colonization. Specific growth rates of 3.79 and 2.83 day−1 were obtained when cells first began to proliferate on a pure chitin film and a silicon surface, respectively. Later, when cell densities on the surface and detached cells as CFU in the bulk aqueous phase achieved a quasi-steady state, specific growth rates decreased to 1.08 and 0.79 day−1 on the chitin and silicon surfaces, respectively. Virtually all of the cells that detached from either the chitin or the silicon surfaces and the majority of cells associated with the chitin surface over the 200-h period of surface colonization displayed no detectable expression of the chitin-degrading genes chiA and chiB. Cells displaying high levels of chiA-chiB expression were detected only on the chitin surface and then only clustered in discrete areas of the surface. Surface-associated, differential gene expression and displacement of bacterial production from surfaces represent adaptations at the population level that promote efficient utilization of limited resources and dispersal of progeny to maximize access to new sources of energy and maintenance of the population. PMID:10919823

  5. Cleavage of metastasis suppressor gene product KiSS-1 protein/metastin by matrix metalloproteinases.

    PubMed

    Takino, Takahisa; Koshikawa, Naohiko; Miyamori, Hisashi; Tanaka, Motohiro; Sasaki, Takuma; Okada, Yasunori; Seiki, Motoharu; Sato, Hiroshi

    2003-07-24

    A human placenta cDNA library was screened by the expression cloning method for gene products that interact with matrix metalloproteinases (MMPs), and we isolated a cDNA whose product formed a stable complex with pro-MMP-2 and pro-MMP-9. The cDNA encoded the metastasis suppressor gene KiSS-1. KiSS-1 protein was shown to form a complex with pro-MMP. KiSS-1 protein is known to be processed to peptide ligand of a G-protein-coupled receptor (hOT7T175) named metastin, and suppresses metastasis of tumors expressing the receptor. Active MMP-2, MMP-9, MT1-MMP, MT3-MMP and MT5-MMP cleaved the Gly118-Leu119 peptide bond of not only full-length KiSS-1 protein but also metastin decapeptide. Metastin decapeptide induced formation of focal adhesion and actin stress fibers in cells expressing the receptor, and digestion of metastin decapeptide by MMP abolished its ligand activity. Migration of HT1080 cells expressing hOT7T175 that harbor a high-level MMP activity was only slightly suppressed by either metastin decapeptide or MMP inhibitor BB-94 alone, but the combination of metastin decapeptide and BB-94 showed a synergistic effect in blocking cell migration. We propose that metastin could be used as an antimetastatic agent in combination with MMP inhibitor, or MMP-resistant forms of metastin could be developed and may also be efficacious.

  6. Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast

    PubMed Central

    Misra, Ashish; Conway, Matthew F.; Johnnie, Joseph; Qureshi, Tabish M.; Lige, Bao; Derrick, Anne M.; Agbo, Eddy C.; Sriram, Ganesh

    2013-01-01

    Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo 13C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. PMID:23898325

  7. Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production.

    PubMed

    Özaydın, Bilge; Burd, Helcio; Lee, Taek Soon; Keasling, Jay D

    2013-01-01

    Beside their essential cellular functions, isoprenoids have value as pharmaceuticals, nutriceuticals, pesticides, and fuel alternatives. Engineering microorganisms for production of isoprenoids is relatively easy, sustainable, and cost effective in comparison to chemical synthesis or extraction from natural producers. We introduced genes encoding carotenoid biosynthetic enzymes into the haploid yeast deletion collection to identify gene deletions that improved isoprenoid production. Deletions that showed significant improvement in carotenoid production were further screened for production of bisabolene, an isoprenoid alternative to petroleum-derived diesel. Combining those deletions with other mevalonate pathway modifications increased production of bisabolene from 40mg/L to 800mg/L in shake-flask cultures. In a fermentation process, this engineered strain produced 5.2g/L of bisabolene.

  8. Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

    PubMed

    Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Wilkins, John A; Cicek, Nazim; Sparling, Richard; Levin, David B

    2015-07-01

    Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

  9. Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product.

    PubMed Central

    Vallone, D; Battista, S; Pierantoni, G M; Fedele, M; Casalino, L; Santoro, M; Viglietto, G; Fusco, A; Verde, P

    1997-01-01

    The expression of the high mobility group I (HMGI)-C chromatin component was shown previously to be essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, we have analyzed the AP-1 complex in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. We show that neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation. PMID:9311991

  10. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    PubMed

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

  11. The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans

    PubMed Central

    Keller, Sebastian; Treder, Aaron; von Reuss, Stephan H.; Escalante-Semerena, Jorge C.

    2016-01-01

    ABSTRACT The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which, in comparison to other cobamides, e.g., cobalamin and pseudo-B12, lacks the methyl group in the linker moiety of the nucleotide loop. In this study, the protein SMUL_1544 was shown to be responsible for the formation of the unusual linker moiety, which is most probably derived from ethanolamine-phosphate (EA-P) as the precursor. The product of the SMUL_1544 gene successfully complemented a Salmonella enterica ΔcobD mutant. The cobD gene encodes an l-threonine-O-3-phosphate (l-Thr-P) decarboxylase responsible for the synthesis of (R)-1-aminopropan-2-ol O-2-phosphate (AP-P), required specifically for cobamide biosynthesis. When SMUL_1544 was produced in the heterologous host lacking CobD, norpseudo-B12 was formed, which pointed toward the formation of EA-P rather than AP-P. Guided cobamide biosynthesis experiments with minimal medium supplemented with l-Thr-P supported cobamide biosynthesis in S. enterica producing SMUL_1544 or S. multivorans. Under these conditions, both microorganisms synthesized pseudo-B12. This observation indicated a flexibility in the SMUL_1544 substrate spectrum. From the formation of catalytically active PCE reductive dehalogenase (PceA) in S. multivorans cells producing pseudo-B12, a compatibility of the respiratory enzyme with the cofactor was deduced. This result might indicate a structural flexibility of PceA in cobamide binding. Feeding of l-[3-13C]serine to cultures of S. multivorans resulted in isotope labeling of the norpseudo-B12 linker moiety, which strongly supports the hypothesis of EA-P formation from l-serine-O-phosphate (l-Ser-P) in this organism. IMPORTANCE The identification of the gene product SMUL_1544 as a putative l-Ser-P decarboxylase involved in norcobamide biosynthesis in S. multivorans adds a novel module to the assembly line of cobamides (complete corrinoids) in prokaryotes

  12. Expression of the transporter encoded by the cefT gene of Acremonium chrysogenum increases cephalosporin production in Penicillium chrysogenum.

    PubMed

    Nijland, Jeroen G; Kovalchuk, Andriy; van den Berg, Marco A; Bovenberg, Roel A L; Driessen, Arnold J M

    2008-10-01

    By introduction of the cefEF genes of Acremonium chrysogenum and the cmcH gene of Streptomyces clavuligerus, Penicillium chrysogenum can be reprogrammed to form adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA), a carbamoylated derivate of adipoyl-7-aminodeacetoxy-cephalosporanic acid. The cefT gene of A. chrysogenum encodes a cephalosporin C transporter that belongs to the Major Facilitator Superfamily. Introduction of cefT into an ad7-ACCCA-producing P. chrysogenum strain results in an almost 2-fold increase in cephalosporin production with a concomitant decrease in penicillin by-product formation. These data suggest that cephalosporin production by recombinant P. chrysogenum strains is limited by the ability of the fungus to secrete these compounds.

  13. Improvement of 1,3-propanediol production using an engineered cyanobacterium, Synechococcus elongatus by optimization of the gene expression level of a synthetic metabolic pathway and production conditions.

    PubMed

    Hirokawa, Yasutaka; Maki, Yuki; Hanai, Taizo

    2017-01-01

    The introduction of a synthetic metabolic pathway consisting of multiple genes derived from various organisms enables cyanobacteria to directly produce valuable chemicals from carbon dioxide. We previously constructed a synthetic metabolic pathway composed of genes from Escherichia coli, Saccharomyces cerevisiae, and Klebsiella pneumoniae. This pathway enabled 1,3-propanediol (1,3-PDO) production from cellular DHAP via glycerol in the cyanobacterium, Synechococcus elongatus PCC 7942. The production of 1,3-PDO (3.79mM, 0.29g/l) directly from carbon dioxide by engineered S. elongatus PCC 7942 was successfully accomplished. However, the constructed strain accumulated a remarkable amount of glycerol (12.6mM, 1.16g/l), an intermediate metabolite in 1,3-PDO production. Notably, enhancement of latter reactions of synthetic metabolic pathway for conversion of glycerol to 1,3-PDO increases 1,3-PDO production. In this study, we aimed to increase the observed 1,3-PDO production titer. First, the weaker S. elongatus PCC 7942 promoter, PLlacO1, was replaced with a stronger promoter (Ptrc) to regulate genes involved in the conversion of glycerol to 1,3-PDO. Second, the induction timing for gene expression and medium composition were optimized. Promoter replacement resulted in higher 1,3-PDO production than glycerol accumulation, and the amount of products (1,3-PDO and glycerol) generated via the synthetic metabolic pathway increased with optimization of medium composition. Accordingly, we achieved the highest titer of 1,3-PDO (16.1mM, 1.22g/l) and this was higher than glycerol accumulation (9.46mM, 0.87g/l). The improved titer was over 4-fold higher than that of our previous study.

  14. Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production.

    PubMed

    Ravanello, Monica P; Ke, Dangyang; Alvarez, Julie; Huang, Bihua; Shewmaker, Christine K

    2003-10-01

    Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed. Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1. This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB. Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B. napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI. The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct. However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with

  15. CONVECTION-ENHANCED DELIVERY AND SYSTEMIC MANNITOL INCREASE GENE PRODUCT DISTRIBUTION OF AAV VECTORS 5, 8, AND 9 AND INCREASE GENE PRODUCT IN THE ADULT MOUSE BRAIN

    PubMed Central

    Carty, Nikisha; Lee, Daniel; Dickey, Chad; Ceballos-Diaz, Carolina; Jansen-West, Karen; Golde, Todd E.; Gordon, Marcia N.; Morgan, Dave; Nash, Kevin

    2010-01-01

    The use of recombinant adeno-associated viral (rAAV) vectors as a means of gene delivery to the central nervous system has emerged as a potentially viable method for the treatment of several types of degenerative brain diseases. However, a limitation of typical intracranial injections into the adult brain parenchyma is the relatively restricted distribution of the delivered gene to large brain regions such as the cortex, presumably due to confined dispersion of the injected particles. Optimizing the administration techniques to maximize gene distribution and gene expression is an important step in developing gene therapy studies. Here, we have found additive increases in distribution when 3 methods to increase brain distribution of rAAV were combined. The convection enhanced delivery (CED) method with the step-design cannula was used to deliver rAAV vector serotypes 5, 8 and 9 encoding GFP into the hippocampus of the mouse brain. While the CED method improved distribution of all 3 serotypes, the combination of rAAV9 and CED was particularly effective. Systemic mannitol administration, which reduces intracranial pressure, also further expanded distribution of GFP expression, in particular, increased expression on the contralateral hippocampi. These data suggest that combining advanced injection techniques with newer rAAV serotypes greatly improves viral vector distribution, which could have significant benefits for implementation of gene therapy strategies. PMID:20951738

  16. Laccase Production and Differential Transcription of Laccase Genes in Cerrena sp. in Response to Metal Ions, Aromatic Compounds, and Nutrients

    PubMed Central

    Yang, Jie; Wang, Guozeng; Ng, Tzi Bun; Lin, Juan; Ye, Xiuyun

    2016-01-01

    Laccases can oxidize a wide range of aromatic compounds and are industrially valuable. Laccases often exist in gene families and may differ from each other in expression and function. Quantitative real-time polymerase chain reaction (qPCR) was used for transcription profiling of eight laccase genes in Cerrena sp. strain HYB07 with validated reference genes. A high laccase activity of 280.0 U/mL was obtained after submerged fermentation for 5 days. Laccase production and laccase gene transcription at different fermentation stages and in response to various environmental cues were revealed. HYB07 laccase activity correlated with transcription levels of its predominantly expressed laccase gene, Lac7. Cu2+ ions were indispensable for efficient laccase production by HYB07, mainly through Lac7 transcription induction, and no aromatic compounds were needed. HYB07 laccase synthesis and biomass accumulation were highest with non-limiting carbon and nitrogen. Glycerol and inorganic nitrogen sources adversely impacted Lac7 transcription, laccase yields, and fungal growth. The present study would further our understanding of transcription regulation of laccase genes, which may in turn facilitate laccase production as well as elucidation of their physiological roles. PMID:26793186

  17. Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product.

    PubMed

    Martin, B; Prats, H; Claverys, J P

    1985-01-01

    The hexA mismatch repair gene of Streptococcus pneumoniae has been cloned into multicopy plasmid vectors. The cloned hexA gene is expressed as judged from its ability to complement various chromosomal hexA- alleles. Its direction of transcription was defined and the functional limits were localized by original methods relying on homology-dependent integration of nonautonomous chimeric plasmids carrying chromosomal inserts into the chromosome. Comparison of the proteins encoded by recombinant plasmids and by restriction fragments allowed us to identify an Mr 94 000 protein as the probable product of the hexA gene.

  18. Elevated CO2 increases photosynthesis, biomass and productivity, and modifies gene expression in sugarcane.

    PubMed

    De Souza, Amanda Pereira; Gaspar, Marilia; Da Silva, Emerson Alves; Ulian, Eugênio César; Waclawovsky, Alessandro Jaquiel; Nishiyama, Milton Yutaka; Dos Santos, Renato Vicentini; Teixeira, Marcelo Menossi; Souza, Glaucia Mendes; Buckeridge, Marcos Silveira

    2008-08-01

    Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric [CO(2)]. The effects of increased [CO(2)] on photosynthesis, development and carbohydrate metabolism were studied in sugarcane (Saccharum ssp.). Plants were grown at ambient (approximately 370 ppm) and elevated (approximately 720 ppm) [CO(2)] during 50 weeks in open-top chambers. The plants grown under elevated CO(2) showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient [CO(2)]. These plants also had lower stomatal conductance and transpiration rates (-37 and -32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO(2). The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher [CO(2)], and that this might be related, as previously observed for maize and sorghum, to transient drought stress.

  19. Heterologous expression of transaldolase gene Tal from Saccharomyces cerevisiae in Fusarium oxysporum for enhanced bioethanol production.

    PubMed

    Fan, Jin-Xia; Yang, Xiao-Xue; Song, Jin-Zhu; Huang, Xiao-Mei; Cheng, Zhong-Xiang; Yao, Lin; Juba, Olivia S; Liang, Qing; Yang, Qian; Odeph, Margaret; Sun, Yan; Wang, Yun

    2011-08-01

    The filamentous fungus Fusarium oxysporum is known for its ability to ferment xylose-producing ethanol. However, efficiency of xylose utilization and ethanol yield was low. In this study, the transaldolase gene from Saccharomyces cerevisiae has been successfully expressed in F. oxysporum by an Agrobacterium tumefaciens-mediated transformation method. The enzymatic activity of the recombinant fungus (cs28pCAM-Sctal4) was 0.195 times higher than that of the wild-type strain (cs28). The recombinant strain also exhibited a 28.83% increase in ethanol yield on xylose media compared to the parental strain. Enhanced ethanol production and a reduction in the biomass were observed during xylose fermentation. Ethanol yield from rice straw by simultaneous saccharification and fermentation with cs28pCAM-Sctal4 was 0.25 g g⁻¹ of rice straw. The transgenic strain of F. oxysporum cs28pCAM-Sctal4 might therefore have potential applications in industrial bioenergy production.

  20. A tissue dependent processing and function of the FMR-1 gene product

    SciTech Connect

    Hoogeveen, A.T.; Verhey, C.; Bakker, C.E.

    1994-09-01

    We recently characterized the FMR-1 gene product (FMRP) associated with the fragile X syndrome. Immunoprecipitation studies were performed on lymphoblastic cells. Four different protein products revealing alternative splicing were found. All these proteins were absent in cells from patients with the fragile X syndrome. Immunolocalization studies on different tissues showed that FMRP was mainly localized in the cytoplasm of the cells. To investigate the possible role and function of the different splice forms, FMRP was isolated from different tissues from human and mouse, using antibodies directed against different parts of the FMRP. RNA binding studies were performed on the isolated forms. For the studies in mouse, tissues from a mouse knockout for FMR-1 were used as a negative control. FMRP isolated from brain, testis, kidney and liver were compared and the results indicate that FMRP is expressed in different forms and ratio in these tissues. The results found in human and mouse were comparable. Further we demonstrated that the different forms of FMRP found for example in brain and kidney have a different affinity for RNA binding.

  1. Norepinephrine causes epigenetic repression of PKCε gene in rodent hearts by activating Nox1-dependent reactive oxygen species production.

    PubMed

    Xiong, Fuxia; Xiao, Daliao; Zhang, Lubo

    2012-07-01

    Heart disease is the leading cause of death in the United States. Recent studies demonstrate that fetal programming of PKCε gene repression results in ischemia-sensitive phenotype in the heart. The present study tests the hypothesis that increased norepinephrine causes epigenetic repression of PKCε gene in the heart via Nox1-dependent reactive oxygen species (ROS) production. Prolonged norepinephrine treatment increased ROS production in fetal rat hearts and embryonic ventricular myocyte H9c2 cells via a selective increase in Nox1 expression. Norepinephrine-induced ROS resulted in an increase in PKCε promoter methylation at Egr-1 and Sp-1 binding sites, leading to PKCε gene repression. N-acetylcysteine, diphenyleneiodonium, and apocynin blocked norepinephrine-induced ROS production and the promoter methylation, and also restored PKCε mRNA and protein to control levels in vivo in fetal hearts and in vitro in embryonic myocyte cells. Accordingly, norepinephrine-induced ROS production, promoter methylation, and PKCε gene repression were completely abrogated by knockdown of Nox1 in cardiomyocytes. These findings provide evidence of a novel interaction between elevated norepinephrine and epigenetic repression of PKCε gene in the heart mediated by Nox1-dependent oxidative stress and suggest new insights of molecular mechanisms linking the heightened sympathetic activity to aberrant cardioprotection and increased ischemic vulnerability in the heart.

  2. Effect of antibiotic down-regulatory gene wblA ortholog on antifungal polyene production in rare actinomycetes Pseudonocardia autotrophica.

    PubMed

    Kim, Hye-Jin; Kim, Min-Kyung; Jin, Ying-Yu; Kim, Young-Woo; Kim, Eung-Soo

    2014-09-01

    The rare actinomycete Pseudonocardia autotrophica was previously shown to produce a solubilityimproved toxicity-reduced novel polyene compound named Nystatin-like Pseudonocardia Polyene (NPP). The low productivity of NPP in P. autotrophica implies that its biosynthetic pathway is tightly regulated. In this study, wblApau was isolated and identified as a novel negative regulatory gene for NPP production in P. autotrophica, which showed approximately 49% amino acid identity with a global antibiotic down-regulatory gene, wblA, identified from various Streptomycetes species. Although no significant difference in NPP production was observed between P. autotrophica harboring empty vector and the S. coelicolor wblA under its native promoter, approximately 12% less NPP was produced in P. autotrophica expressing the wblA gene under the strong constitutive ermE(*) promoter. Furthermore, disruption of the wblApau gene from P. autotrophica resulted in an approximately 80% increase in NPP productivity. These results strongly suggest that identification and inactivation of the global antibiotic down-regulatory gene wblA ortholog are a critical strategy for improving secondary metabolite overproduction in not only Streptomyces but also non-Streptomyces rare actinomycete species.

  3. Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli.

    PubMed

    Liu, Xiang-Lei; Lin, Jun; Hu, Hai-Feng; Zhou, Bin; Zhu, Bao-Quan

    2016-04-01

    Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).

  4. Maternal obesity in the agouti viable yellow (Avy) mouse produces defective secretory activation that is associated with mammary inflammation and activation of adrenocorticosteroid-dependent gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maternal obesity is known to interfere with normal lactation in women, rodents, and dairy animals. Obesity is also correlated with profound changes in an array of endocrine factors and is causally linked with inflammation and insulin resistance. Recent work suggests that elevated aldosterone actin...

  5. Overexpression of D-xylose reductase (xyl1) gene and antisense inhibition of D-xylulokinase (xyiH) gene increase xylitol production in Trichoderma reesei.

    PubMed

    Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

    2014-01-01

    T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

  6. Recent advances in awakening silent biosynthetic gene clusters and linking orphan clusters to natural products in microorganisms.

    PubMed

    Chiang, Yi-Ming; Chang, Shu-Lin; Oakley, Berl R; Wang, Clay C C

    2011-02-01

    Secondary metabolites from microorganisms have a broad spectrum of applications, particularly in therapeutics. The growing number of sequenced microbial genomes has revealed a remarkably large number of natural product biosynthetic clusters for which the products are still unknown. These cryptic clusters are potentially a treasure house of medically useful compounds. The recent development of new methodologies has made it possible to begin unlock this treasure house, to discover new natural products and to determine their biosynthesis pathways. This review will highlight some of the most recent strategies to activate silent biosynthetic gene clusters and to elucidate their corresponding products and pathways.

  7. Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis.

    PubMed

    Inoue, Yasushi; Yasutake, Nozomu; Oshima, Yoshie; Yamamoto, Yoshie; Tomita, Tetsuji; Miyoshi, Shinsuke; Yatake, Tsuneya

    2002-12-01

    The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.

  8. 1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon

    SciTech Connect

    I-Teh Tong; Hans H. Liao; Cameron, D.C. )

    1991-12-01

    The dha regulon in Klebsiella pneumoniae enables the organism to grown anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydoxyacetone and was screened for the production of 1, 3-PD. The cosmid pTC1 (42.5 kn total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycersol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1, 3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.

  9. FlhA influences Bacillus thuringiensis PlcR-regulated gene transcription, protein production, and virulence.

    PubMed

    Bouillaut, Laurent; Ramarao, Nalini; Buisson, Christophe; Gilois, Nathalie; Gohar, Michel; Lereclus, Didier; Nielsen-Leroux, Christina

    2005-12-01

    Bacillus thuringiensis and Bacillus cereus are closely related. B. thuringiensis is well known for its entomopathogenic properties, principally due to the synthesis of plasmid-encoded crystal toxins. B. cereus appears to be an emerging opportunistic human pathogen. B. thuringiensis and B. cereus produce many putative virulence factors which are positively controlled by the pleiotropic transcriptional regulator PlcR. The inactivation of plcR decreases but does not abolish virulence, indicating that additional factors like flagella may contribute to pathogenicity. Therefore, we further analyzed a mutant (B. thuringiensis 407 Cry(-) DeltaflhA) previously described as being defective in flagellar apparatus assembly and in motility as well as in the production of hemolysin BL and phospholipases. A large picture of secreted proteins was obtained by two-dimensional electrophoresis analysis, which revealed that flagellar proteins are not secreted and that production of several virulence-associated factors is reduced in the flhA mutant. Moreover, we quantified the effect of FlhA on plcA and hblC gene transcription. The results show that the flhA mutation results in a significant reduction of plcA and hblC transcription. These results indicate that the transcription of several PlcR-regulated virulence factors is coordinated with the flagellar apparatus. Consistently, the flhA mutant also shows a strong decrease in cytotoxicity towards HeLa cells and in virulence against Galleria mellonella larvae following oral and intrahemocoelic inoculation. The decrease in virulence may be due to both a lack of flagella and a lower production of secreted factors. Hence, FlhA appears to be an essential virulence factor with a pleiotropic role.

  10. Discovery of Gene Sources for Economic Traits in Hanwoo by Whole-genome Resequencing

    PubMed Central

    Shin, Younhee; Jung, Ho-jin; Jung, Myunghee; Yoo, Seungil; Subramaniyam, Sathiyamoorthy; Markkandan, Kesavan; Kang, Jun-Mo; Rai, Rajani; Park, Junhyung; Kim, Jong-Joo

    2016-01-01

    Hanwoo, a Korean native cattle (Bos taurus coreana), has great economic value due to high meat quality. Also, the breed has genetic variations that are associated with production traits such as health, disease resistance, reproduction, growth as well as carcass quality. In this study, next generation sequencing technologies and the availability of an appropriate reference genome were applied to discover a large amount of single nucleotide polymorphisms (SNPs) in ten Hanwoo bulls. Analysis of whole-genome resequencing generated a total of 26.5 Gb data, of which 594,716,859 and 592,990,750 reads covered 98.73% and 93.79% of the bovine reference genomes of UMD 3.1 and Btau 4.6.1, respectively. In total, 2,473,884 and 2,402,997 putative SNPs were discovered, of which 1,095,922 (44.3%) and 982,674 (40.9%) novel SNPs were discovered against UMD3.1 and Btau 4.6.1, respectively. Among the SNPs, the 46,301 (UMD 3.1) and 28,613 SNPs (Btau 4.6.1) that were identified as Hanwoo-specific SNPs were included in the functional genes that may be involved in the mechanisms of milk production, tenderness, juiciness, marbling of Hanwoo beef and yellow hair. Most of the Hanwoo-specific SNPs were identified in the promoter region, suggesting that the SNPs influence differential expression of the regulated genes relative to the relevant traits. In particular, the non-synonymous (ns) SNPs found in CORIN, which is a negative regulator of Agouti, might be a causal variant to determine yellow hair of Hanwoo. Our results will provide abundant genetic sources of variation to characterize Hanwoo genetics and for subsequent breeding. PMID:26954201

  11. Identification of the Genes Involved in the Fruiting Body Production and Cordycepin Formation of Cordyceps militaris Fungus

    PubMed Central

    Zheng, Zhuang-li; Qiu, Xue-hong

    2015-01-01

    A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris. PMID:25892913

  12. The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes

    SciTech Connect

    Yamano, Shigeru; Tatsuno, Jun; Gonzalez, F.J. )

    1990-02-06

    Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a {lambda}gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu{sup 160}{yields}His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a {lambda}{sub max} at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIa mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. These data establish that the CYP2A3 gene product is primarily responsible for coumarin 7-hydroxylase activity in human liver. The level of expression of this activity varied up to 40-fold between livers. Levels of IIA mRNA also varied significantly between liver specimens, and three specimens had no detectable mRNA.

  13. Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product.

    PubMed

    Himmelfarb, H J; Maicas, E; Friesen, J D

    1985-04-01

    The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known.

  14. Pistil Transcriptome Analysis to Disclose Genes and Gene Products Related to Aposporous Apomixis in Hypericum perforatum L.

    PubMed Central

    Galla, Giulio; Zenoni, Sara; Avesani, Linda; Altschmied, Lothar; Rizzo, Paride; Sharbel, Timothy F.; Barcaccia, Gianni

    2017-01-01

    Unlike sexual reproduction, apomixis encompasses a number of reproductive strategies, which permit maternal genome inheritance without genetic recombination and syngamy. The key biological features of apomixis are the circumvention of meiosis (i.e., apomeiosis), the differentiation of unreduced embryo sacs and egg cells, and their autonomous development in functional embryos through parthenogenesis, and the formation of viable endosperm either via fertilization-independent means or following fertilization with a sperm cell. Despite the importance of apomixis for breeding of crop plants and although much research has been conducted to study this process, the genetic control of apomixis is still not well understood. Hypericum perforatum is becoming an attractive model system for the study of aposporous apomixis. Here we report results from a global gene expression analysis of H. perforatum pistils collected from sexual and aposporous plant accessions for the purpose of identifying genes, biological processes and molecular functions associated with the aposporous apomixis pathway. Across two developmental stages corresponding to the expression of aposporous apomeiosis and parthenogenesis in ovules, a total of 224 and 973 unigenes were found to be significantly up- and down-regulated with a fold change ≥ 2 in at least one comparison, respectively. Differentially expressed genes were enriched for multiple gene ontology (GO) terms, including cell cycle, DNA metabolic process, and single-organism cellular process. For molecular functions, the highest scores were recorded for GO terms associated with DNA binding, DNA (cytosine-5-)-methyltransferase activity and heterocyclic compound binding. As deregulation of single components of the sexual developmental pathway is believed to be a trigger of the apomictic reproductive program, all genes involved in sporogenesis, gametogenesis and response to hormonal stimuli were analyzed in great detail. Overall, our data suggest that

  15. Pistil Transcriptome Analysis to Disclose Genes and Gene Products Related to Aposporous Apomixis in Hypericum perforatum L.

    PubMed

    Galla, Giulio; Zenoni, Sara; Avesani, Linda; Altschmied, Lothar; Rizzo, Paride; Sharbel, Timothy F; Barcaccia, Gianni

    2017-01-01

    Unlike sexual reproduction, apomixis encompasses a number of reproductive strategies, which permit maternal genome inheritance without genetic recombination and syngamy. The key biological features of apomixis are the circumvention of meiosis (i.e., apomeiosis), the differentiation of unreduced embryo sacs and egg cells, and their autonomous development in functional embryos through parthenogenesis, and the formation of viable endosperm either via fertilization-independent means or following fertilization with a sperm cell. Despite the importance of apomixis for breeding of crop plants and although much research has been conducted to study this process, the genetic control of apomixis is still not well understood. Hypericum perforatum is becoming an attractive model system for the study of aposporous apomixis. Here we report results from a global gene expression analysis of H. perforatum pistils collected from sexual and aposporous plant accessions for the purpose of identifying genes, biological processes and molecular functions associated with the aposporous apomixis pathway. Across two developmental stages corresponding to the expression of aposporous apomeiosis and parthenogenesis in ovules, a total of 224 and 973 unigenes were found to be significantly up- and down-regulated with a fold change ≥ 2 in at least one comparison, respectively. Differentially expressed genes were enriched for multiple gene ontology (GO) terms, including cell cycle, DNA metabolic process, and single-organism cellular process. For molecular functions, the highest scores were recorded for GO terms associated with DNA binding, DNA (cytosine-5-)-methyltransferase activity and heterocyclic compound binding. As deregulation of single components of the sexual developmental pathway is believed to be a trigger of the apomictic reproductive program, all genes involved in sporogenesis, gametogenesis and response to hormonal stimuli were analyzed in great detail. Overall, our data suggest that

  16. Providencia stuartii genes activated by cell-to-cell signaling and identification of a gene required for production or activity of an extracellular factor.

    PubMed

    Rather, P N; Ding, X; Baca-DeLancey, R R; Siddiqui, S

    1999-12-01

    By utilizing reporter transposons, five Providencia stuartii genes that are activated by the accumulation of self-produced extracellular signals have been identified. These genes have been designated cma for conditioned medium activated. The presence of conditioned medium from stationary-phase cultures grown in rich media resulted in the premature activation of each gene in cells at early log phase, with activation values ranging from 6- to 26-fold. Preparation of conditioned medium from an M9 salts medium and fractionation by gel filtration chromatography resulted in fractions within the included volume which activated three of the cma fusions. In addition, depending on the reporter fusion, peak activity was found in different fractions. The partially purified factors activated in a dose-dependent manner. Characterization of the factors activating the cma fusions indicated that they were stable to heat, alkali, and acid. Furthermore, for each cma fusion, factor activity was not reproduced by the addition of homoserine lactone, homocysteine thiolactone, pyruvate, Casamino Acids, or alpha-ketoglutarate. The identities of three cma genes have been determined and revealed physiological roles in amino acid biosynthesis and nutrient import. To begin to address the pathways for production of or response to the extracellular factors, we have identified a locus, aarA, that is required for the activation of four cma fusions. The AarA product was required for factor activity in extracellular supernatants, indicating a possible role in biosynthesis or export.

  17. Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues.

    PubMed

    Cordon-Cardo, C; O'Brien, J P; Boccia, J; Casals, D; Bertino, J R; Melamed, M R

    1990-09-01

    We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp

  18. Genes encoding the production of extracellular polysaccharide bioflocculant are clustered on a 30-kb DNA segment in Bacillus licheniformis.

    PubMed

    Yan, Shan; Wang, Na; Chen, Zhen; Wang, Yuanpeng; He, Ning; Peng, Yajuan; Li, Qingbiao; Deng, Xu

    2013-11-01

    Bioflocculants are special high-molecular weight polymers produced by microorganisms. Despite the fact that several types of bioflocculants from different species of bacteria have been reported, there is a large gap in our knowledge regarding the molecular machine responsible for the production of bioflocculants. To investigate genes involved in bioflocculant synthesis, a fosmid library was generated from Bacillus licheniformis genomic DNA and screened for the production of bioflocculant. Four positive clones with distinct flocculation were isolated by a two-pooling scheme. The clone with 662 U ml(-1) flocculating activity was sequenced. As a result, a 30-kb fragment with 26 hypothetical genes was identified in the bioflocculant-producing clone. Most of the predicted proteins encoded by the inserted genes showed significant homology with enzymes involved in the biosynthesis of polysaccharide. Based on these homologies, a biosynthesis pathway and two gene clusters involved in the production of the polysaccharide bioflocculant were proposed with the integration of functional descriptions of individual genes by metabolic databases, and a glucose-sensitive glycosidases was predicted. This research supplied significant data for potential application of bioflocculant-producing strains in wastewater refining and industrial downstream treatments.

  19. Dynamic Linkages between Denitrification Functional Genes/Enzymes and Biogeochemical Reaction Rates of Nitrate and Its Reduction Products

    NASA Astrophysics Data System (ADS)

    Li, M.; Shi, L.; Qian, W.; Gao, Y.; Liu, Y.; Liu, C.

    2015-12-01

    Denitrification is a respiratory process in which oxidized nitrogen compounds are used as alternative electron acceptors for energy production when oxygen is limited. Denitrification is an important process that not only accounts for the significant loss of nitrogen fertilizers from soils but also leads to NO, N2O and CO2 emissions, which are important greenhouse gas species. In this study, denitrification was investigated in Columbia River sediments, focusing on the dynamic linkages between functional genes/enzymes and biogeochemical reaction rates of nitrate and its reduction products. NO3-, NO2- and N2O were assayed in different incubation time. DNA was extracted from the sediments and functional genes were quantified as a function of time during the denitrification. Functional enzymes were extracted from the sediments and measured using a newly developed, targeted protein method. The biogeochemical, functional gene, and enzyme data were collectively used to establish the dynamic correlation of functional genes/enzymes and biogeochemical reaction rates. The results provide fundamental insights regarding the dynamic regulation of functional genes and enzymes in the processes of denitrification and greenhouse gas production, and also provide experimental data critical for the development of biogeochemical reaction models that incorporate genome-scale insights and describe macroscopic biogeochemical reaction rates in ecosystems.

  20. Increased Production of Fatty Acids and Triglycerides in Aspergillus oryzae by Enhancing Expressions of Fatty Acid Synthesis-Related Genes

    SciTech Connect

    Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.; Culley, David E.; Deng, Shuang; Collett, James R.; Umemura, Myco; Koike, Hideaki; Baker, Scott E.; Machida, Masa

    2013-01-01

    Microbial production of fats and oils is being developedas a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillusoryzae. Examination of the A.oryzaegenome demonstrates that it contains twofatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhancedthe expressionof fatty acid synthesis-related genes by replacing their promoters with thepromoter fromthe constitutively highly expressedgene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthasegenes we successfullyincreasedtheproduction of fatty acids and triglyceridesby more than two fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesteraseincreasedproductivity to a lesser extent.Increasing expression ofacetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored usingquantitative real-time RT-PCR. Our data demonstrates that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

  1. Wounding tomato fruit elicits ripening-stage specific changes in gene expression and production of volatile compounds.

    PubMed

    Baldassarre, Valentina; Cabassi, Giovanni; Spadafora, Natasha D; Aprile, Alessio; Müller, Carsten T; Rogers, Hilary J; Ferrante, Antonio

    2015-03-01

    Fleshy fruits develop from an unripe organ that needs to be protected from damage to a ripe organ that attracts frugivores for seed dispersal through production of volatile organic compounds (VOCs). Thus, different responses to wounding damage are predicted. The aim of this study was to discover whether wound-induced changes in the transcriptome and VOC production alter as tomato transitions from unripe to ripe. Transcript changes were analysed 3h post-wounding using microarray analysis in two commercial salad-tomato (Solanum lycopersicum L.) cultivars: Luna Rossa and AVG, chosen for their high aroma production. This was followed by quantitative PCR on Luna Rossa genes involved in VOC biosynthesis and defence responses. VOCs elicited by wounding at different ripening stages were analysed by solid phase micro extraction and gas chromatography-mass spectrometry. Approximately 4000 differentially expressed genes were identified in the cultivar AVG and 2500 in Luna Rossa. In both cultivars the majority of genes were up-regulated and the most affected pathways were metabolism of terpenes, carotenoids, and lipids. Defence-related genes were mostly up-regulated in immature stages of development, whereas expression of genes related to VOCs changed at riper stages. More than 40 VOCs were detected and profiles changed with ripening stage. Thus, both transcriptome and VOC profiles elicited by wounding depend on stage of ripening, indicating a shift from defence to attraction.

  2. The Global Regulatory hns Gene Negatively Affects Adhesion to Solid Surfaces by Anaerobically Grown Escherichia coli by Modulating Expression of Flagellar Genes and Lipopolysaccharide Production

    PubMed Central

    Landini, Paolo; Zehnder, Alexander J. B.

    2002-01-01

    The initial binding of bacterial cells to a solid surface is a critical and essential step in biofilm formation. In this report we show that stationary-phase cultures of Escherichia coli W3100 (a K-12 strain) can efficiently attach to sand columns when they are grown in Luria broth medium at 28°C in fully aerobic conditions. In contrast, growth in oxygen-limited conditions results in a sharp decrease in adhesion to hydrophilic substrates. We show that the production of lipopolysaccharide (LPS) and of flagella, as well as the transcription of the fliC gene, encoding the major flagellar subunit, increases under oxygen-limited conditions. Inactivation of the global regulatory hns gene counteracts increased production of LPS and flagella in response to anoxia and allows E. coli W3100 to attach to sand columns even when it is grown under oxygen-limited conditions. We propose that increased production of the FliC protein and of LPS in response to oxygen limitation results in the loss of the ability of E. coli W3100 to adhere to hydrophilic surfaces. Indeed, overexpression of the fliC gene results in a decreased adhesion to sand even when W3100 is grown in fully aerobic conditions. Our observations strongly suggest that anoxia is a negative environmental signal for adhesion in E. coli. PMID:11872702

  3. The Natural Product Domain Seeker NaPDoS: A Phylogeny Based Bioinformatic Tool to Classify Secondary Metabolite Gene Diversity

    PubMed Central

    Ziemert, Nadine; Podell, Sheila; Penn, Kevin; Badger, Jonathan H.; Allen, Eric; Jensen, Paul R.

    2012-01-01

    New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry. PMID:22479523

  4. Functional screening yields a new beta-1,4-endoglucanase gene from Heterodera glycines that may be the product of recent gene duplication.

    PubMed

    Yan, Y; Smant, G; Davis, E

    2001-01-01

    Clones with secreted cellulolytic activity were identified when a cDNA library constructed from poly A(+) RNA of preparasitic second-stage juveniles of Heterodera glycines, the soybean cyst nematode, was expressed in the Escherichia coli SOLR strain and overlaid with a carboxymethylcellulose (CMC) substrate. Twenty CMC-degrading clones were analyzed, and all were either identical or strongly similar to a beta-1,4-endoglucanase gene (HG-eng-2), previously isolated from H. glycines. A subgroup of identical "HG-eng-2-like" clones had considerable differences in the 5' untranslated region compared with HG-eng-2 and were designated HG-eng-3. One H. glycines genomic clone contained HG-eng-2 and HG-eng-3 full-length genes, separated by a distance of approximately 8 kb, and a second genomic clone contained two copies of HG-eng-2, separated by approximately 6.5 kb, suggesting the presence of endoglucanase gene clusters in H. glycines. The HG-eng-2 and HG-eng-3 genes were in opposite transcriptional orientation, with considerable nucleotide differences in their 5' flanking regions. The highly conserved nucleotide sequence in the introns and exons and their close proximity within the genome suggest that HG-eng-2 and HG-eng-3 are the products of recent gene duplication and inversion.

  5. Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene.

    PubMed

    Li, Huan-Jun; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2016-01-10

    Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation.

  6. Exploring the Transcriptome Landscape of Pomegranate Fruit Peel for Natural Product Biosynthetic Gene and SSR Marker Discovery(F).

    PubMed

    Ono, Nadia Nicole; Britton, Monica Therese; Fass, Joseph Nathaniel; Nicolet, Charles Meyer; Lin, Dawei; Tian, Li

    2011-10-01

    Pomegranate fruit peel is rich in bioactive plant natural products, such as hydrolyzable tannins and anthocyanins. Despite their documented roles in human nutrition and fruit quality, genes involved in natural product biosynthesis have not been cloned from pomegranate and very little sequence information is available on pomegranate in the public domain. Shotgun transcriptome sequencing of pomegranate fruit peel cDNA was performed using RNA-Seq on the Illumina Genome Analyzer platform. Over 100 million raw sequence reads were obtained and assembled into 9,839 transcriptome assemblies (TAs) (>200 bp). Candidate genes for hydrolyzable tannin, anthocyanin, flavonoid, terpenoid and fatty acid biosynthesis and/or regulation were identified. Three lipid transfer proteins were obtained that may contribute to the previously reported IgE reactivity of pomegranate fruit extracts. In addition, 115 SSR markers were identified from the pomegranate fruit peel transcriptome and primers were designed for 77 SSR markers. The pomegranate fruit peel transcriptome set provides a valuable platform for natural product biosynthetic gene and SSR marker discovery in pomegranate. This work also demonstrates that next-generation transcriptome sequencing is an economical and effective approach for investigating natural product biosynthesis, identifying genes controlling important agronomic traits, and discovering molecular markers in non-model specialty crop species.

  7. Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

    PubMed

    Romanczuk, H; Wormington, W M

    1989-02-01

    Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

  8. The common fragile site FRA16D gene product WWOX: roles in tumor suppression and genomic stability.

    PubMed

    Aqeilan, Rami I; Abu-Remaileh, Muhannad; Abu-Odeh, Mohammad

    2014-12-01

    The fragile WWOX gene, encompassing the chromosomal fragile site FRA16D, is frequently altered in human cancers. While vulnerable to DNA damage itself, recent evidence has shown that the WWOX protein is essential for proper DNA damage response (DDR). Furthermore, the gene product, WWOX, has been associated with multiple protein networks, highlighting its critical functions in normal cell homeostasis. Targeted deletion of Wwox in murine models suggests its in vivo requirement for proper growth, metabolism, and survival. Recent molecular and biochemical analyses of WWOX functions highlighted its role in modulating aerobic glycolysis and genomic stability. Cumulatively, we propose that the gene product of FRA16D, WWOX, is a functionally essential protein that is required for cell homeostasis and that its deletion has important consequences that contribute to the neoplastic process. This review discusses the essential role of WWOX in tumor suppression and genomic stability and how its alteration contributes to cancer transformation.

  9. Elucidating the Rimosamide-Detoxin Natural Product Families and Their Biosynthesis Using Metabolite/Gene Cluster Correlations.

    PubMed

    McClure, Ryan A; Goering, Anthony W; Ju, Kou-San; Baccile, Joshua A; Schroeder, Frank C; Metcalf, William W; Thomson, Regan J; Kelleher, Neil L

    2016-12-16

    As microbial genome sequencing becomes more widespread, the capacity of microorganisms to produce an immense number of metabolites has come into better view. Utilizing a metabolite/gene cluster correlation platform, the biosynthetic origins of a new family of natural products, the rimosamides, were discovered. The rimosamides were identified in Streptomyces rimosus and associated with their NRPS/PKS-type gene cluster based upon their high frequency of co-occurrence across 179 strains of actinobacteria. This also led to the discovery of the related detoxin gene cluster. The core of each of these families of natural products contains a depsipeptide bond at the point of bifurcation in their unusual branched structures, the origins of which are definitively assigned to nonlinear biosynthetic pathways via heterologous expression in Streptomyces lividans. The rimosamides were found to antagonize the antibiotic activity of blasticidin S against Bacillus cereus.

  10. Variation in Siderophore Biosynthetic Gene Distribution and Production across Environmental and Faecal Populations of Escherichia coli

    PubMed Central

    Porcelli, Ida; Sheppard, Samuel K.; Lucchini, Sacha

    2015-01-01

    Iron is essential for Escherichia coli growth and survival in the host and the external environment, but its availability is generally low due to the poor solubility of its ferric form in aqueous environments and the presence of iron-withholding proteins in the host. Most E. coli can increase access to iron by excreting siderophores such as enterobactin, which have a very strong affinity for Fe3+. A smaller proportion of isolates can generate up to 3 additional siderophores linked with pathogenesis; aerobactin, salmochelin, and yersiniabactin. However, non-pathogenic E. coli are also able to synthesise these virulence-associated siderophores. This raises questions about their role in the ecology of E. coli, beyond virulence, and whether specific siderophores might be linked with persistence in the external environment. Under the assumption that selection favours phenotypes that confer a fitness advantage, we compared siderophore production and gene distribution in E. coli isolated either from agricultural plants or the faeces of healthy mammals. This population-level comparison has revealed that under iron limiting growth conditions plant-associated isolates produced lower amounts of siderophores than faecal isolates. Additionally, multiplex PCR showed that environmental isolates were less likely to contain loci associated with aerobactin and yersiniabactin synthesis. Although aerobactin was linked with strong siderophore excretion, a significant difference in production was still observed between plant and faecal isolates when the analysis was restricted to strains only able to synthesise enterobactin. This finding suggests that the regulatory response to iron limitation may be an important trait associated with adaptation to the non-host environment. Our findings are consistent with the hypothesis that the ability to produce multiple siderophores facilitates E. coli gut colonisation and plays an important role in E. coli commensalism. PMID:25756870

  11. Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

    DOE PAGES

    Dupont, Chris L.; McCrow, John P.; Valas, Ruben; ...

    2014-10-21

    Here, transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enrichedmore » in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.« less

  12. Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

    SciTech Connect

    Dupont, Chris L.; McCrow, John P.; Valas, Ruben; Moustafa, Ahmed; Walworth, Nathan; Goodenough, Ursula; Roth, Robyn; Hogle, Shane L.; Bai, Jing; Johnson, Zackary I.; Mann, Elizabeth; Palenik, Brian; Barbeau, Katherine A.; Venter, J. Craig; Allen, Andrew E.

    2014-10-21

    Here, transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.

  13. Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

    PubMed Central

    Dupont, Chris L; McCrow, John P; Valas, Ruben; Moustafa, Ahmed; Walworth, Nathan; Goodenough, Ursula; Roth, Robyn; Hogle, Shane L; Bai, Jing; Johnson, Zackary I; Mann, Elizabeth; Palenik, Brian; Barbeau, Katherine A; Craig Venter, J; Allen, Andrew E

    2015-01-01

    Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM. PMID:25333462

  14. Variation in siderophore biosynthetic gene distribution and production across environmental and faecal populations of Escherichia coli.

    PubMed

    Searle, Laura J; Méric, Guillaume; Porcelli, Ida; Sheppard, Samuel K; Lucchini, Sacha

    2015-01-01

    Iron is essential for Escherichia coli growth and survival in the host and the external environment, but its availability is generally low due to the poor solubility of its ferric form in aqueous environments and the presence of iron-withholding proteins in the host. Most E. coli can increase access to iron by excreting siderophores such as enterobactin, which have a very strong affinity for Fe3+. A smaller proportion of isolates can generate up to 3 additional siderophores linked with pathogenesis; aerobactin, salmochelin, and yersiniabactin. However, non-pathogenic E. coli are also able to synthesise these virulence-associated siderophores. This raises questions about their role in the ecology of E. coli, beyond virulence, and whether specific siderophores might be linked with persistence in the external environment. Under the assumption that selection favours phenotypes that confer a fitness advantage, we compared siderophore production and gene distribution in E. coli isolated either from agricultural plants or the faeces of healthy mammals. This population-level comparison has revealed that under iron limiting growth conditions plant-associated isolates produced lower amounts of siderophores than faecal isolates. Additionally, multiplex PCR showed that environmental isolates were less likely to contain loci associated with aerobactin and yersiniabactin synthesis. Although aerobactin was linked with strong siderophore excretion, a significant difference in production was still observed between plant and faecal isolates when the analysis was restricted to strains only able to synthesise enterobactin. This finding suggests that the regulatory response to iron limitation may be an important trait associated with adaptation to the non-host environment. Our findings are consistent with the hypothesis that the ability to produce multiple siderophores facilitates E. coli gut colonisation and plays an important role in E. coli commensalism.

  15. Segregation of yeast polymorphic STA genes in meiotic recombinants and analysis of glucoamylase production.

    PubMed

    Balogh, I; Maráz, A

    1996-12-01

    Hybrid yeast strains were constructed using haploid Saccharomyces cerevisiae and Saccharomyces cerevisiae var. diastaticus strains to get haploid meiotic recombinants having more than one copy of STA1, STA2, and STA3 genes. STA genes were localized on the chromosomes by pulsed field gel electrophoresis. Working gene dosage effects were found among STA genes in liquid starch medium, indicating low levels of glucose repression. Growth of strains, however, was not influenced by their STA copy number.

  16. Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product.

    PubMed Central

    Gassmann, G S; Jacobs, E; Deutzmann, R; Göbel, U B

    1991-01-01

    The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis. Images PMID:1704884

  17. Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle

    PubMed Central

    2013-01-01

    Background Identification of single nucleotide polymorphisms (SNPs) for specific genes involved in reproduction might improve reliability of genomic estimates for these low-heritability traits. Semen from 550 Holstein bulls of high (≥ 1.7; n = 288) or low (≤ −2; n = 262) daughter pregnancy rate (DPR) was genotyped for 434 candidate SNPs using the Sequenom MassARRAY® system. Three types of SNPs were evaluated: SNPs previously reported to be associated with reproductive traits or physically close to genetic markers for reproduction, SNPs in genes that are well known to be involved in reproductive processes, and SNPs in genes that are differentially expressed between physiological conditions in a variety of tissues associated in reproductive function. Eleven reproduction and production traits were analyzed. Results A total of 40 SNPs were associated (P < 0.05) with DPR. Among these were genes involved in the endocrine system, cell signaling, immune function and inhibition of apoptosis. A total of 10 genes were regulated by estradiol. In addition, 22 SNPs were associated with heifer conception rate, 33 with cow conception rate, 36 with productive life, 34 with net merit, 23 with milk yield, 19 with fat yield, 13 with fat percent, 19 with protein yield, 22 with protein percent, and 13 with somatic cell score. The allele substitution effect for SNPs associated with heifer conception rate, cow conception rate, productive life and net merit were in the same direction as for DPR. Allele substitution effects for several SNPs associated with production traits were in the opposite direction as DPR. Nonetheless, there were 29 SNPs associated with DPR that were not negatively associated with production traits. Conclusion SNPs in a total of 40 genes associated with DPR were identified as well as SNPs for other traits. It might be feasible to include these SNPs into genomic tests of reproduction and other traits. The genes associated with DPR are likely to be

  18. Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker.

    PubMed

    Khan, Raham Sher; Ntui, Valentine Otang; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro

    2011-04-01

    The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.

  19. Molecular Networking and Pattern-Based Genome Mining Improves Discovery of Biosynthetic Gene Clusters and their Products from Salinispora Species

    SciTech Connect

    Duncan, Katherine R.; Crüsemann, Max; Lechner, Anna; Sarkar, Anindita; Li, Jie; Ziemert, Nadine; Wang, Mingxun; Bandeira, Nuno; Moore, Bradley S.; Dorrestein, Pieter C.; Jensen, Paul R.

    2015-04-09

    Genome sequencing has revealed that bacteria contain many more biosynthetic gene clusters than predicted based on the number of secondary metabolites discovered to date. While this biosynthetic reservoir has fostered interest in new tools for natural product discovery, there remains a gap between gene cluster detection and compound discovery. In this paper, we apply molecular networking and the new concept of pattern-based genome mining to 35 Salinispora strains, including 30 for which draft genome sequences were either available or obtained for this study. The results provide a method to simultaneously compare large numbers of complex microbial extracts, which facilitated the identification of media components, known compounds and their derivatives, and new compounds that could be prioritized for structure elucidation. Finally, these efforts revealed considerable metabolite diversity and led to several molecular family-gene cluster pairings, of which the quinomycin-type depsipeptide retimycin A was characterized and linked to gene cluster NRPS40 using pattern-based bioinformatic approaches.

  20. Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

    PubMed Central

    2013-01-01

    Background Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important

  1. Characterization of Erwinia chrysanthemi PY35 cel and pel gene existing in tandem and rapid identification of their gene products.

    PubMed

    Park, S R; Kim, M K; Kim, J O; Bae, D W; Cho, S J; Cho, Y U; Yun, H D

    2000-02-16

    Genomic DNA of the phytopathogenic Erwinia chrysanthemi PY35 was partially digested with Sau3AI, ligated into the BamHI site of pBluescript II SK+, and introduced into E. coli. One clone that was able to hydrolyse carboxymethylcellulose and polygalacturonic acid was selected. A 2.9 kb fragment containing the pelL1 gene (pPY300) and cel5Z gene (pPY401) in tandem was subcloned and sequenced. The pelL1 and cel5Z genes had open reading frames of 1,278 bp and 1,281 bp encoding 425 and 426 amino acid residues with calculated molecular weights of 45,649 Da and 46,473 Da, respectively. pelL1 and cel5Z carried a typical prokaryotic signal peptide of 24 and 41 amino acid residues, respectively. The apparent molecular masses of the proteins when expressed in E. coli cells were approximately 43 kDa (PelL1) and 42 kDa (Cel5Z) as assessed by PGA-SDS-PAGE and CMC-SDS-PAGE.

  2. Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase.

    PubMed Central

    Lin, B T; Gruenwald, S; Morla, A O; Lee, W H; Wang, J Y

    1991-01-01

    The retinoblastoma gene product (RB) is a nuclear protein which has been shown to function as a tumor suppressor. It is phosphorylated from S to M phase of the cell cycle and dephosphorylated in G1. This suggests that the function of RB is regulated by its phosphorylation in the cell cycle. Ten phosphotryptic peptides are found in human RB proteins. The pattern of RB phosphorylation does not change from S to M phases of the cell cycle. Hypophosphorylated RB prepared from insect cells infected with an RB-recombinant baculovirus is used as a substrate for in vitro phosphorylation reactions. Of several protein kinases tested, only cdc2 kinase phosphorylates RB efficiently and all 10 peptides can be phosphorylated by cdc2 in vitro. Removal of cdc2 from mitotic cell extracts by immunoprecipitation causes a concomitant depletion of RB kinase activity. These results indicate that cdc2 or a kinase with similar substrate specificity is involved in the cell cycle-dependent phosphorylation of the RB protein. Images PMID:2009861

  3. Cardiovascular actions of DOPA mediated by the gene product of ocular albinism 1.

    PubMed

    Goshima, Yoshio; Nakamura, Fumio; Masukawa, Daiki; Chen, Sandy; Koga, Motokazu

    2014-01-01

    l-3,4-Dihydroxyphenylalanine (DOPA) is the metabolic precursor of dopamine, and the single most effective agent in the treatment of Parkinson's disease. One problem with DOPA therapy for Parkinson's disease is its cardiovascular side effects including hypotension and syncope, the underlying mechanisms of which are largely unknown. We proposed that DOPA is a neurotransmitter in the central nervous system, but specific receptors for DOPA had not been identified. Recently, the gene product of ocular albinism 1 (OA1) was shown to possess DOPA-binding activity. It was unknown, however, whether or not OA1 is responsible for the actions of DOPA itself. Immunohistochemical examination revealed that OA1 was expressed in the nucleus tractus solitarii (NTS). OA1-positive cells adjacent to tyrosine hydroxylase-positive cell bodies and nerve fibers were detected in the depressor sites of the NTS. OA1 knockdown using oa1-specific shRNA-adenovirus vectors in the NTS reduced the expression levels of OA1 in the NTS. The prior injection of the shRNA against OA1 suppressed the depressor and bradycardic responses to DOPA but not to glutamate in the NTS of anesthetized rats. Thus OA-1 is a functional receptor of DOPA in the NTS, which warrants reexamination of the mechanisms for the therapeutic and untoward actions of DOPA.

  4. Localization of ocular albinism-1 gene product GPR143 in the rat central nervous system.

    PubMed

    Masukawa, Daiki; Nakamura, Fumio; Koga, Motokazu; Kamiya, Marina; Chen, Sandy; Yamashita, Naoya; Arai, Nobutaka; Goshima, Yoshio

    2014-11-01

    L-3,4-Dihydroxyphenylalanine (DOPA) has been believed to be a precursor of dopamine, and itself being an inert amino acid. Previously, we have proposed DOPA as a neurotransmitter candidate in the central nervous system (CNS). Recent findings have suggested DOPA as an endogenous agonist of a G-protein coupled receptor, ocular albinism 1 gene product (OA1), which is highly expressed in the retinal pigmental epithelium. However, whether OA1 functions as a receptor for DOPA in vivo, and whether this receptor-ligand interaction is responsible for a wide variety of DOPA actions have not been determined yet. To gain insight into the functional implication of OA1, we perform immunohistochemical examination with anti-OA1 antibody to localize OA1 in the adult rat brain. We observed OA1 immunoreactive cells in the hippocampus, cerebral cortex, cerebellum cortex, striatum, substantia nigra, hypothalamic median eminence and supraoptic nucleus, nucleus tractus solitarii and caudal ventrolateral medulla and rostral ventrolateral medulla, medial habenular nucleus and olfactory bulb. This study reveals, for the first time, the unique distribution pattern of OA1-immunoreactive neurons and/or cells in the rat CNS.

  5. The distribution and expression of the Bloom's syndrome gene product in normal and neoplastic human cells.

    PubMed

    Turley, H; Wu, L; Canamero, M; Gatter, K C; Hickson, I D

    2001-07-20

    Bloom's syndrome (BS) is an autosomal recessive disorder associated with a predisposition to cancers of all types. Cells from BS sufferers display extreme genomic instability. The BS gene product, BLM, is a 159 kDa DNA helicase enzyme belonging to the RecQ family. Here, we have analysed the distribution of BLM in normal and tumour tissues from humans using a recently characterized, specific monoclonal antibody. BLM was found to be localized to nuclei in normal lymphoid tissues, but was largely absent from other normal tissues analysed with the exception of the proliferating compartment of certain tissues. In contrast, expression of BLM was observed in a variety of tumours of both lymphoid and epithelial origin. A strong correlation was observed between expression of BLM and the proliferative status of cells, as determined by staining for markers of cell proliferation (PCNA and Ki67). We conclude that BLM is a proliferation marker in normal and neoplastic cells in vivo, and, as a consequence, is expressed at a higher level in tumours than in normal quiescent tissues.

  6. PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases.

    PubMed

    Ullah, Abul H J; Sethumadhavan, Kandan

    2003-04-04

    PhyA gene products of Aspergillus ficuum (AF) and Peniophora lycii (PL) as expressed in industrial strains of Aspergillus niger and Aspergillus oryzae, respectively, were purified to homogeneity and then characterized for both physical and biochemical properties. The PL phytase is 26 amino acid residues shorter than the AF phytase. Dynamic light scattering studies indicate that the active AF phytase is a monomer while the PL phytase is a dimer. While both of the phytases retained four identical glycosylatable Asn residues, unique glycosylation sites, six for PL and seven for AF phytase, were observed. Global alignment of both the phytases has shown 38% sequence homology between the two proteins. At 58 degrees C and pH 5.0, the PL phytase gave a specific activity of 22,000 nKat/mg as opposed to about 3000 nKat/mg for AF phytase. However, the AF phytase is more thermostable than its counterpart PL phytase at 65 degrees C. Also, AF phytase is more stable at pH 7.5 than the PL phytase. The two phytases differed in K(m) for phytate, K(i) for myo-inositol hexasulfate (MIHS), and pH optima profile. Despite similarities in the active site sequences, the two phytases show remarkable differences in turnover number, pH optima profile, stability at higher temperature, and alkaline pH. These biochemical differences indicate that phytases from ascomycete and basidiomycete fungi may have evolved to degrade phytate in different environments.

  7. Regulation of transcription of the adenovirus EII promoter by gene products: Absence of sequence specificity

    SciTech Connect

    Kingston, R.E.; Kaufman, R.J.; Sharp, P.A.

    1984-10-01

    During adenovirus infection, the EII promoter is positively regulated by products of the EIa region. The authors have studied this regulation by fusing a DNA segment containing the adenovirus EII promoter to a dihydrofolate reductase cDNA segment. Expression of this hybrid gene is stimulated in trans when cell lines containing an integrated copy are either transfected with plasmids carrying the EIa region or infected with adenovirus. This suggests that EIa activity regulates transcription of the EII promoter in the absence of other viral proteins and that this stimulation can occur when the EII promoter is organized in cellular chromatin. Transcription from the EII promoter is initiated at two sites in cell lines lacking EIa activity. Introduction of the EIa region preferentially stimulated transcription from one of these two sites. A sensitive, stable cotransfection assay was used to test for specific EII sequences required for stimulation. EIa activity stimulates all mutaant promoters; the most extensive deletion retained only 18 base pairs of sequences upstream of the initiation site. They suggest that regulation of a promoter by the EIa region does not depend on the presence of a set of specific sequences, but instead reflects a characteristic of promoters that have been exogenously introduced into cells. Insertion of the 72-base-pair repeat of simian-virus 40 in cis enhances transcription from the EII promoter. The stimulatory effects of EIa activity and of the simian virus 40 sequence are additive and appear to differ mechanistically.

  8. Differential sorting and packaging of capa-gene related products in an insect.

    PubMed

    Pollák, Edit; Eckert, Manfred; Molnár, László; Predel, Reinhard

    2005-01-03

    A unique costorage of neuropeptides was recently found in the abdominal perisympathetic organs (PSOs) of the American cockroach, Periplaneta americana. Having specific antisera directed against all peptides belonging to this neurosecretory system, we examined the sorting of PSO-peptides in the soma of the median neurosecretory cells of abdominal ganglia by using immunoelectron microscopic double stainings. The data indicate that all six abundant neuropeptides of this neurohormonal system, which includes three capa-gene related products, are primarily incorporated into separate vesicles. These vesicles fuse with each other in the cytoplasm and become translucent on their way to the axon hillock. By means of light microscopy and MALDI-TOF mass spectrometry, an identical population of neuropeptides was found in interneurons of the brain. As revealed by subsequent immunoelectron microscopic analysis, the peptides of these cells are separately packed into dense core vesicles but do not fuse with each other. Thus, hitherto unknown cell-type-specific sorting mechanisms occur in neurosecretory cells and interneurons, respectively.

  9. Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

    PubMed Central

    Klessig, D F; Brough, D E; Cleghon, V

    1984-01-01

    The DNA-binding protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central role in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by DBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DBP is toxic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50- to 200-fold, and within 8 h its synthesis from the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells. Images PMID:6542172

  10. Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

    PubMed

    Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

    2016-09-01

    Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production.

  11. Optimizing Transgene Configuration and Protein Fusions to Maximize Dopamine Production for the Gene Therapy of Parkinson's Disease.

    PubMed

    Stewart, Hannah J; Ralph, G Scott; Fong-Wong, Liang; Strickland, Iain; McCloskey, Laura; Barnes, Lucy; Blount, Ian; Wells, Owen; Truran, Christelle J M; Kingsman, Alan J; Palfi, Stéphane; Mitrophanous, Kyriacos A

    2016-09-01

    Pharmacological dopamine replacement therapies provide the most well-established treatments for Parkinson's disease (PD). However, these long-term treatments can lead to motor complications and off-target effects. ProSavin(®), a lentiviral vector (LV)-based gene therapy approach aimed at restoring local and continuous dopamine production, through delivery of three enzymes in the dopamine biosynthesis pathway, was demonstrated to be safe and well-tolerated in a phase I/II clinical study of patients with advanced PD. Although improvements in motor behaviour were observed, the data indicated that higher levels of dopamine replacement might be required to maximize benefit. We attempted to increase production of dopamine, and its precursor L-Dopa in LV-transduced cells, by optimizing the gene order in the ProSavin expression cassette, and by creating fusions of two or three of the transgenes, using linker sequences. In vitro analysis showed that several gene arrangements provided significantly increased dopamine and/or L-Dopa production compared with ProSavin, and that LV titers and transgene expression were not affected by introducing gene fusions. One vector, equine infectious anemia virus (EIAV)-TCiA, was selected for further characterization and showed significant improvements in dopamine and L-Dopa production compared with ProSavin, in human neuronal cells. Further characterization of EIAV-TCiA demonstrated expression of all three dopamine enzymes in vivo and faithful delivery and integration of the expected gene expression cassette within the genome of target cells, as assessed by Northern and Southern blotting. In conclusion, we have developed a novel LV vector with an increased capacity for L-Dopa and dopamine production compared with the current ProSavin vector. Clinical evaluation of this vector will be performed to assess the benefits in patients with PD.

  12. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

  13. Enhanced cyanophycin production by Escherichia coli overexpressing the heterologous cphA gene from a deep sea metagenomic library.

    PubMed

    Du, Jikun; Li, Li; Zhou, Shining

    2017-02-01

    Cyanophycin is a non-ribosomally synthesized polymer and its microbial production has attracted increased attention due to its pharmaceutical and chemical values. For the characterization and production of cyanophycin, the cphA49 gene was cloned and expressed in Escherichia coli. Soluble cyanophycin was isolated from the cultures and characterized. The results showed that it was composed of 50% of aspartic acid, 45% of arginine, and 3.5% of lysine, and exhibited a homogenous molecular mass of 35 kDa. To improve soluble cyanophycin production, the induction conditions for cphA49 gene expression were optimized. Meanwhile, the effects of medium content and induction duration on soluble cyanophycin production were also investigated, and the soluble cyanophycin yield reached a maximum at 72 h. Finally, to further increase the soluble cyanophycin production, the cultivation was carried out by supplement of arginine, aspartic acid, lysine and glucose into the minimal resource medium after cphA49 gene expression level was improved under optimized conditions, and the maximum concentration of soluble cyanophycin reached 1.72 g/L.

  14. Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

    PubMed

    Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

    2015-02-01

    Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus amyloliquefaciens LL3. Products of rocR, rocG and gudB facilitate the conversion from glutamate to 2-oxoglutarate in Bacillus subtillis. The gene odhA is responsible for the synthesis of a component of the 2-oxoglutarate dehydrogenase complex that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A. In-frame deletions of these four genes were performed. In shake flask experiments the gudB/rocG double mutant presented enhanced production of γ-PGA, a 38 % increase compared with wild type. When fermented in a 5-L fermenter with pH control, the γ-PGA yield of the rocR mutant was increased to 5.83 g/L from 4.55 g/L for shake flask experiments. The gudB/rocG double mutant produced 5.68 g/L γ-PGA compared with that of 4.03 g/L for the wild type, a 40 % increase. Those results indicated the possibility of improving γ-PGA production by modifying glutamate metabolism, and identified potential genetic targets to improve γ-PGA production.

  15. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  16. Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures

    PubMed Central

    Lijavetzky, Diego; Almagro, Lorena; Belchi-Navarro, Sarai; Martínez-Zapater, José M; Bru, Roque; Pedreño, Maria A

    2008-01-01

    Background Plant cell cultures have been shown as feasible systems for the production of secondary metabolites, being the elicitation with biotic or abiotic stimuli the most efficient strategy to increase the production of those metabolites. Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family which are derivatives of the trans-resveratrol structure and are produced by plants and cell cultures as a response to biotic and abiotic stresses. The potential benefits of resveratrol on human health have made it one of the most thoroughly studied phytochemical molecules. The aim of this study was to evaluate the elicitor effect of both cyclodextrin (CD) and methyljasmonate (MeJA) on grapevine cell cultures by carrying out a quantitative analysis of their role on resveratrol production and on the expression of stilbene biosynthetic genes in Vitis vinifera cv Monastrell albino cell suspension cultures. Findings MeJA and CD significantly but transiently induced the expression of stilbene biosynthetic genes when independently used to treat grapevine cells. This expression correlated with resveratrol production in CD-treated cells but not in MeJA-treated cells, which growth was drastically affected. In the combined treatment of CD and MeJA cell growth was similarly affected, however resveratrol production was almost one order of magnitude higher, in correlation with maximum expression values for stilbene biosynthetic genes. Conclusion The effect of MeJA on cell division combined with a true and strong elicitor like CD could be responsible for the observed synergistic effect of both compounds on resveratrol production and on the expression of genes in the stilbene pathway. PMID:19102745

  17. Microarray analysis of changes in cellular gene expression induced by productive infection of primary human astrocytes: implications for HAD.

    PubMed

    Kim, Seon-Young; Li, Jinliang; Bentsman, Galina; Brooks, Andrew I; Volsky, David J

    2004-12-01

    The role of astrocytes in HIV-1 associated dementia (HAD) is not well understood. HIV-1 binds efficiently to astrocytes but infects only a small fraction of the cells in vitro and in vivo. To gain insight into the biology of HIV-1-expressing astrocytes, we productively infected human fetal astrocytes with pseudotyped HIV-1 and employed Affymetrix oligonucleotide microarrays to determine global changes in cellular gene expression at the peak of virus production. With a twofold change as a cutoff, HIV-1 increased transcription of 266 genes in astrocytes and suppressed expression of 468. The functions of highly expressed genes included interferon-mediated antiviral responses (OAS1, IFIT1), intercellular contacts (SH3, glia-derived nexin), cell homing/adhesion (matrix metalloproteinases), and cell-cell signaling (neuropilin 1 and 2). Surprisingly, genes involved in innate immune responses of astrocytes were largely unaffected. The single most significant effect of HIV-1, however, was down-modulation of at least 55 genes involved in control of cell cycle, DNA replication, and cell proliferation, which were overrepresented in these categories with probability scores of 10(-10)-10(-26). Our data suggest that HIV-1 expression in astrocytes profoundly alters host cell biology, with potential consequences for the physiological function of astrocytes during HIV-1 infection in the brain.

  18. Product of Saccharomyces cerevisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA.

    PubMed Central

    Costanzo, M C; Fox, T D

    1986-01-01

    The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII). Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-beta-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria. To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion. These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII. The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs. These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA. Images PMID:3099165

  19. Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus.

    PubMed

    Moyano, Elisabet; Jouhikainen, Katja; Tammela, Päivi; Palazón, Javier; Cusidó, Rosa M; Piñol, M Teresa; Teeri, Teemu H; Oksman-Caldentey, Kirsi-Marja

    2003-01-01

    In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.

  20. Nebulon: a system for the inference of functional relationships of gene products from the rearrangement of predicted operons

    PubMed Central

    Janga, Sarath Chandra; Collado-Vides, Julio; Moreno-Hagelsieb, Gabriel

    2005-01-01

    Since operons are unstable across Prokaryotes, it has been suggested that perhaps they re-combine in a conservative manner. Thus, genes belonging to a given operon in one genome might re-associate in other genomes revealing functional relationships among gene products. We developed a system to build networks of functional relationships of gene products based on their organization into operons in any available genome. The operon predictions are based on inter-genic distances. Our system can use different kinds of thresholds to accept a functional relationship, either related to the prediction of operons, or to the number of non-redundant genomes that support the associations. We also work by shells, meaning that we decide on the number of linking iterations to allow for the complementation of related gene sets. The method shows high reliability benchmarked against knowledge-bases of functional interactions. We also illustrate the use of Nebulon in finding new members of regulons, and of other functional groups of genes. Operon rearrangements produce thousands of high-quality new interactions per prokaryotic genome, and thousands of confirmations per genome to other predictions, making it another important tool for the inference of functional interactions from genomic context. PMID:15867197

  1. Detection and Diversity Evaluation of Tetracycline Resistance Genes in Grassland-Based Production Systems in Colombia, South America

    PubMed Central

    Santamaría, Johanna; López, Liliana; Soto, Carlos Yesid

    2011-01-01

    Grassland-based production systems use ∼26% of land surface on earth. However, there are no evaluations of these systems as a source of antibiotic pollution. This study was conducted to evaluate the presence, diversity, and distribution of tetracycline resistance genes in the grasslands of the Colombian Andes, where administration of antibiotics to animals is limited to treat disease and growth promoters are not included in animals’ diet. Animal (ruminal fluid and feces) and environmental (soil and water) samples were collected from different dairy cattle farms and evaluated by PCR for the genes tet(M), tet(O), tetB(P), tet(Q), tet(W), tet(S), tet(T), otr(A), which encode ribosomal protection proteins (RPPs), and the genes tet(A), tet(B), tet(D), tet(H), tet(J), and tet(Z), encoding efflux pumps. A wide distribution and high frequency for genes tet(W) and tet(Q) were found in both sample types. Genes tet(O) and tetB(P), detected in high frequencies in feces, were detected in low frequencies or not detected at all in the environment. Other genes encoding RPPs, such as tet(M), tet(S), and tet(T), were detected at very low frequencies and restricted distributions. Genes encoding efflux pumps were not common in this region, and only two of them, tet(B) and tet(Z), were detected. DGGE–PCR followed by comparative sequence analysis of tet(W) and tet(Q) showed that the sequences detected in animals did not differ from those coming from soil and water. Finally, the farms sampled in this study showed more than 50% similarity in relation to the tet genes detected. In conclusion, there was a remarkable presence of tet genes in these production systems and, although not all genes detected in animal reservoirs were detected in the environment, there is a predominant distribution of tet(W) and tet(Q) in both animal and environmental reservoirs. Sequence similarity analysis suggests the transmission of these genes from animals to the environment. PMID:22174707

  2. Structure, Expression, Chromosomal Location and Product of the Gene Encoding Adh2 in Petunia

    PubMed Central

    Gregerson, R. G.; Cameron, L.; McLean, M.; Dennis, P.; Strommer, J.

    1993-01-01

    In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes. PMID:8096485

  3. SxtA and sxtG Gene Expression and Toxin Production in the Mediterranean Alexandrium minutum (Dinophyceae)

    PubMed Central

    Perini, Federico; Galluzzi, Luca; Dell’Aversano, Carmela; Dello Iacovo, Emma; Tartaglione, Luciana; Ricci, Fabio; Forino, Martino; Ciminiello, Patrizia; Penna, Antonella

    2014-01-01

    The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions. PMID:25341029

  4. Comparative Genomic Analysis of Delftia tsuruhatensis MTQ3 and the Identification of Functional NRPS Genes for Siderophore Production

    PubMed Central

    Guo, Haimeng; Yang, Yanan; Liu, Kai; Xu, Wenfeng; Gao, Jianyong; Duan, Hairong; Du, Binghai

    2016-01-01

    Plant growth-promoting rhizobacteria (PGPR) are a group of rhizosphere bacteria that promote plant growth. Delftia tsuruhatensis MTQ3 is a member of PGPR that produces siderophores. The draft genome sequence of MTQ3 has been reported. Here, we analyzed the genome sequence of MTQ3 and performed a comparative genome analysis of four sequenced Delftia strains, revealing genetic relationships among these strains. In addition, genes responsible for bacteriocin and nonribosomal peptide synthesis were detected in the genomes of each strain. To reveal the functions of NRPS genes in siderophore production in D. tsuruhatensis MTQ3, three NRPS genes were knocked out to obtain the three mutants MTQ3-Δ1941, MTQ3-Δ1945, and MTQ3-Δ1946, which were compared with the wild-type strain. In qualitative and quantitative analyses using CAS assay, the mutants failed to produce siderophores. Accordingly, the NRPS genes in MTQ3 were functionally related to siderophore production. These results clarify one mechanism by which plant growth is promoted in MTQ3 and have important applications in agricultural production. PMID:27847812

  5. Four novel polymorphisms of buffalo INSIG2 gene are associated with milk production traits in Chinese buffaloes.

    PubMed

    Deng, Tingxian; Pang, Chunying; Ma, Xiaoya; Lu, Xingrong; Duan, Anqin; Zhu, Peng; Liang, Xianwei

    2016-10-01

    Insulin-induced genes (INSIGs), including INSIG1 and INSIG2, are important mediators that play a pivotal role in the lipid metabolism and could cause the retention of the SCAP/SREBP complex. Therefore, the objective of this study is to detect the single nucleotide polymorphisms (SNPs) of buffalo INSIG2 gene and evaluate their associations with milk production traits in Chinese buffaloes. A total of four SNPs (g.621272A > G, g.621364A > C, g.632543G > A, and g.632684C > T) were identified using DNA pooled sequencing, and the SNP genotyping for the identified SNPs was performed by using Matrix-assisted laser desorption/ionization time of flight mass spectrometry method from 264 individuals. The results showed that four SNPs were significantly associated with 305-day milk yield or protein percentage in Murrah and crossbred breeds (P < 0.05), but they had no significant effect on milk production traits in Nili-Ravi buffaloes (P > 0.05). Linkage disequilibrium (LD) analysis revealed that one haplotype block was successfully constructed, of which the diplotype H1H1 showed significant association with 305-day milk yield in Murrah buffaloes (P < 0.05). Our findings provide evidence that polymorphisms in buffalo INSIG2 gene are associated with milk production traits, and could be used as a candidate gene for marker-assisted selection in buffalo breeding program.

  6. Comparative Genomic Analysis of Delftia tsuruhatensis MTQ3 and the Identification of Functional NRPS Genes for Siderophore Production.

    PubMed

    Guo, Haimeng; Yang, Yanan; Liu, Kai; Xu, Wenfeng; Gao, Jianyong; Duan, Hairong; Du, Binghai; Ding, Yanqin; Wang, Chengqiang

    2016-01-01

    Plant growth-promoting rhizobacteria (PGPR) are a group of rhizosphere bacteria that promote plant growth. Delftia tsuruhatensis MTQ3 is a member of PGPR that produces siderophores. The draft genome sequence of MTQ3 has been reported. Here, we analyzed the genome sequence of MTQ3 and performed a comparative genome analysis of four sequenced Delftia strains, revealing genetic relationships among these strains. In addition, genes responsible for bacteriocin and nonribosomal peptide synthesis were detected in the genomes of each strain. To reveal the functions of NRPS genes in siderophore production in D. tsuruhatensis MTQ3, three NRPS genes were knocked out to obtain the three mutants MTQ3-Δ1941, MTQ3-Δ1945, and MTQ3-Δ1946, which were compared with the wild-type strain. In qualitative and quantitative analyses using CAS assay, the mutants failed to produce siderophores. Accordingly, the NRPS genes in MTQ3 were functionally related to siderophore production. These results clarify one mechanism by which plant growth is promoted in MTQ3 and have important applications in agricultural production.

  7. Use of Staby(®) technology for development and production of DNA vaccines free of antibiotic resistance gene.

    PubMed

    Reschner, Anca; Scohy, Sophie; Vandermeulen, Gaëlle; Daukandt, Marc; Jacques, Céline; Michel, Benjamin; Nauwynck, Hans; Xhonneux, Florence; Préat, Véronique; Vanderplasschen, Alain; Szpirer, Cédric

    2013-10-01

    The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby(®) technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby(®) technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1).

  8. Association of single nucleotide polymorphism in melatonin receptor 1A gene with egg production traits in Yangzhou geese.

    PubMed

    Alsiddig, M A; Yu, S G; Pan, Z X; Widaa, H; Badri, T M; Chen, J; Liu, H L

    2017-04-01

    In the present study the melatonin receptor 1A gene (MTNR1A) was proposed to be a candidate gene for egg production in Yangzhou geese. A total of 210 goose blood samples were collected to investigate the association of the MTNR1A gene with the number of eggs produced. Using a direct sequencing method, a single nucleotide polymorphism (SNP; g.177G>C) was detected in the 5' regulatory region of the MTNR1A gene (Genbank ss1985399687). Two alleles (G and C) and three genotypes were identified. Association analysis results showed that the g.177G>C SNP significantly affected the level of egg production within a 34-week egg-laying period (P < 0.05). Furthermore, the geese with the GG genotype produced significantly more eggs compared to the geese with the CC genotype. Quantitative real-time PCR analysis showed that the MTNR1A gene was highly expressed in small intestine, granulosa cell and ovary compared to other examined tissues. In addition, the mRNA expression level of MTNR1A in ovary indicated that significantly higher expression levels were recorded for geese with the GG genotype compared to those with the CC genotype. Moreover, a luciferase reporter assay showed that the CC genotype had significantly lower promoter activity than did GG. These results suggest that the identified SNP in the MTNR1A gene may influence the number of eggs produced and mRNA expression levels in Yangzhou geese and could be considered as a useful molecular marker in goose selection and improvement, especially for egg production.

  9. Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example

    PubMed Central

    Taniguchi, Hironori; Wendisch, Volker F.

    2015-01-01

    Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production. PMID:26257719

  10. Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example.

    PubMed

    Taniguchi, Hironori; Wendisch, Volker F

    2015-01-01

    Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production.

  11. Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environmentally active compounds

    SciTech Connect

    Gracia, Tannia Hilscherova, Klara; Jones, Paul D.; Newsted, John L.; Higley, Eric B.; Zhang, Xiaowei; Hecker, Markus; Murphy, Margaret B.; Yu, Richard M.K.; Lam, Paul K.S.; Wu, Rudolf S.S.; Giesy, John P.

    2007-12-01

    The H295R cell bioassay was used to evaluate the potential endocrine disrupting effects of 18 of the most commonly used pharmaceuticals in the United States. Exposures for 48 h with single pharmaceuticals and binary mixtures were conducted; the expression of five steroidogenic genes, 3{beta}HSD2, CYP11{beta}1, CYP11{beta}2, CYP17 and CYP19, was quantified by Q-RT-PCR. Production of the steroid hormones estradiol (E2), testosterone (T) and progesterone (P) was also evaluated. Antibiotics were shown to modulate gene expression and hormone production. Amoxicillin up-regulated the expression of CYP11{beta}2 and CYP19 by more than 2-fold and induced estradiol production up to almost 3-fold. Erythromycin significantly increased CYP11{beta}2 expression and the production of P and E2 by 3.5- and 2.4-fold, respectively, while production of T was significantly decreased. The {beta}-blocker salbutamol caused the greatest induction of CYP17, more than 13-fold, and significantly decreased E2 production. The binary mixture of cyproterone and salbutamol significantly down-regulated expression of CYP19, while a mixture of ethynylestradiol and trenbolone, increased E2 production 3.7-fold. Estradiol production was significantly affected by changes in concentrations of trenbolone, cyproterone, and ethynylestradiol. Exposures with individual pharmaceuticals showed the possible secondary effects that drugs may exert on steroid production. Results from binary mixture exposures suggested the possible type of interactions that may occur between drugs and the joint effects product of such interactions. Dose-response results indicated that although two chemicals may share a common mechanism of action the concentration effects observed may be significantly different.

  12. High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

    PubMed Central

    Catone, Mariela V.; Ruiz, Jimena A.; Castellanos, Mildred; Segura, Daniel; Espin, Guadalupe; López, Nancy I.

    2014-01-01

    Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases. PMID:24887088

  13. Evidence for Horizontal Gene Transfer as Origin of Putrescine Production in Oenococcus oeni RM83▿

    PubMed Central

    Marcobal, Ángela; de las Rivas, Blanca; Moreno-Arribas, M. Victoria; Muñoz, Rosario

    2006-01-01

    The nucleotide sequence of a 17.2-kb chromosomal DNA fragment containing the odc gene encoding ornithine decarboxylase has been determined in the putrescine producer Oenococcus oeni RM83. This DNA fragment contains 13 open reading frames, including genes coding for five transposases and two phage proteins. This description might represent the first evidence of a horizontal gene transfer event as the origin of a biogenic amine biosynthetic locus. PMID:17056681

  14. Production of Truncated Candida antarctica Lipase B Gene Using Automated PCR Gene Assembly Protocol and Expression in Yeast for use in Ethanol and Biodiesel Production.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An improved column-based process for production of biodiesel was developed using a column containing a strongly basic anion-exchange resin in sequence with a column containing a resin to which a lipase biocatalyst is bound. Currently most biodiesel is produced by transesterification of triglyceride...

  15. The effect of neurospecific knockdown of candidate genes for locomotor behavior and sound production in Drosophila melanogaster.