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Sample records for human amylase genes

  1. Expression of the human amylase genes: Recent origin of a salivary amylase promoter from an actin pseudogene

    SciTech Connect

    Samuelson, L.C.; Gumucio, D.L.; Meisler, M.H. ); Wiebauer, K. )

    1988-09-12

    The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. The authors have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B, is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5{prime} regions of the five human amylase genes contain a processed {gamma}-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3{prime} untranslated region of {gamma}-actin. In addition, insertion of an endogenous retrovirus has interrupted the {gamma}-actin pseudogene in four of the five amylase genes.

  2. Expression of the human amylase genes: recent origin of a salivary amylase promoter from an actin pseudogene.

    PubMed

    Samuelson, L C; Wiebauer, K; Gumucio, D L; Meisler, M H

    1988-09-12

    The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. We have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B, is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5' regions of the five human amylase genes contain a processed gamma-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3' untranslated region of gamma-actin. In addition, insertion of an endogenous retrovirus has interrupted the gamma-actin pseudogene in four of the five amylase genes. PMID:2458567

  3. Cloning and characterization of a third type of human alpha-amylase gene, AMY2B.

    PubMed

    Yokouchi, H; Horii, A; Emi, M; Tomita, N; Doi, S; Ogawa, M; Mori, T; Matsubara, K

    1990-06-15

    We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes. PMID:2401405

  4. Diet and the evolution of human amylase gene copy number variation

    PubMed Central

    Perry, George H.; Dominy, Nathaniel J.; Claw, Katrina G.; Lee, Arthur S.; Fiegler, Heike; Redon, Richard; Werner, John; Villanea, Fernando A.; Mountain, Joanna L.; Misra, Rajeev; Carter, Nigel P.; Lee, Charles; Stone, Anne C.

    2008-01-01

    Starch consumption is a prominent characteristic of agricultural societies and hunter-gatherers in arid environments. In contrast, rainforest and circum-arctic hunter-gatherers and some pastoralists consume much less starch1-3. This behavioral variation raises the possibility that different selective pressures have acted on amylase, the enzyme responsible for starch hydrolysis4. We found that salivary amylase gene (AMY1) copy number is correlated positively with salivary amylase protein levels, and that individuals from populations with high-starch diets have on average more AMY1 copies than those with traditionally low-starch diets. Comparisons with other loci in a subset of these populations suggest that the level of AMY1 copy number differentiation is unusual. This example of positive selection on a copy number variable gene is one of the first in the human genome. Higher AMY1 copy numbers and protein levels likely improve the digestion of starchy foods, and may buffer against the fitness-reducing effects of intestinal disease. PMID:17828263

  5. Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes

    PubMed Central

    Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M.; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A.A.; Yang, Fengtang; Thomas, Mark G.; Armour, John A.L.

    2015-01-01

    The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. PMID:25788522

  6. Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes.

    PubMed

    Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A A; Yang, Fengtang; Thomas, Mark G; Armour, John A L

    2015-06-15

    The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. PMID:25788522

  7. Alpha-amylase gene transcription in tissues of normal dog.

    PubMed

    Mocharla, H; Mocharla, R; Hodes, M E

    1990-02-25

    We studied the distribution of alpha-amylase mRNA in normal dog tissues by northern blotting (NB) and reverse transcription-polymerase chain reaction (RT-PCR) with human pancreatic (AMY2) and salivary (AMY1) alpha-amylase cDNA-specific primers. Analysis of poly(A+) RNA from various normal tissues by NB indicated the presence of detectable levels of alpha-amylase mRNA transcripts only in pancreas. Dot-blot analysis of DNA amplified with primers common to both (human) isoamylase mRNAs showed presence of alpha-amylase gene transcripts not only in pancreas but also in liver, small intestine, large intestine and fallopian tube. Traces of amylase gene transcripts were also observed in ovary, uterus and lung. Interestingly, amylase transcripts were not detectable in the parotid gland by NB or RT-PCR. We have also localized alpha-amylase mRNA transcripts to dog pancreas by in situ transcription and in situ hybridization. Our results suggest that there is high degree of homology between the alpha-amylase mRNA sequences in dog and human at least in the exon 3-4 regions of the human gene. PMID:2315015

  8. α-Amylase structural genes in rye.

    PubMed

    Masojć, P; Gale, M D

    1991-10-01

    Rye α-Amy1, α-Amy2, and α-Amy3 genes were studied in the cross between inbred lines using wheat α-amylase cDNA probes. The α-Amy1 and α-Amy2 probes uncovered considerable restriction fragment length polymorphism, whereas the α-Amy3 region was much more conserved. The numbers of restriction fragments found and the F2 segregation data suggest that there are three α-Amy1 genes, two or three α-Amy2 genes, and three α-Amy3 genes in rye. These conclusions were supported by a simultaneous study of α-amylase isozyme polymorphism. The F2 data showed the three individual α-Amy1 genes to span a distance of 3cM at the locus on chromosome 6RL. The genes were mapped relative to other RFLP markers on 6RL. On chromosome 7RL two α-Amy2 genes were shown to be separated by 5 cM. Linkage data within α-Amy3 on 5RL were not obtained since RFLP could be detected at only one of the genes. PMID:24213454

  9. A novel type of human alpha-amylase produced in lung carcinoid tumor.

    PubMed

    Tomita, N; Horii, A; Doi, S; Yokouchi, H; Shiosaki, K; Higashiyama, M; Matsuura, N; Ogawa, M; Mori, T; Matsubara, K

    1989-03-15

    A novel type of alpha-amylase was detected in a lung carcinoid tissue after surveying the cDNA library constructed from this tumor mRNA. Nucleotide sequence analysis showed that the amylase expressed in this carcinoid tumor has 13 and 6 amino acid substitutions when compared with salivary amylase (Amy1) and pancreatic amylase (Amy2), respectively. The nucleotide sequence homologies of cDNAs between this carcinoid amylase and amy1, amy2 are 97.5% and 98.2%, respectively. The nucleotide sequence comparison strongly suggests that this new amylase is the product of the amy3 gene that has been detected in human genome [Emi et al., Gene 62 (1988) 229-235] PMID:2701942

  10. Classification and evolution of alpha-amylase genes in plants.

    PubMed

    Huang, N; Stebbins, G L; Rodriguez, R L

    1992-08-15

    The DNA sequences for 17 plant genes for alpha-amylase (EC 3.2.1.1) were analyzed to determine their phylogenetic relationship. A phylogeny for these genes was obtained using two separate approaches, one based on molecular clock assumptions and the other based on a comparison of sequence polymorphisms (i.e., small and localized insertions) in the alpha-amylase genes. These polymorphisms are called "alpha-amylase signatures" because they are diagnostic of the gene subfamily to which a particular alpha-amylase gene belongs. Results indicate that the cereal alpha-amylase genes fall into two major classes: AmyA and AmyB. The AmyA class is subdivided into the Amy1 and Amy2 subfamilies previously used to classify alpha-amylase genes in barley and wheat. The AmyB class includes the Amy3 subfamily to which most of the alpha-amylase genes of rice belong. Using polymerase chain reaction and oligonucleotide primers that flank one of the two signature regions, we show that the AmyA and AmyB gene classes are present in approximately equal amounts in all grass species examined except barley. The AmyB (Amy3 subfamily) genes in the latter case are comparatively underrepresented. Additional evidence suggests that the AmyA genes appeared recently and may be confined to the grass family. PMID:1502164

  11. Location of the alpha-amylase gene in rumen Streptococcus bovis strains distinguished by unstable amylase activity.

    PubMed

    Mareková, M; Jonecová, Z; Kmeĭ, V

    1995-01-01

    Genetic stability of amylase activity after serial subcultivation experiments with amylolytic ruminal Streptococcus bovis strains was investigated. Two strains Amy+ and Amy- were obtained. Loss of amylase activity connected with the loss of plasmid DNA was not found in these strains. The presence of the gene responsible for the amylase activity in the chromosome of these strains was revealed by hybridization of the alpha-amylase gene on pJK108 against chromosomal DNA of S. bovis and Bacillus subtilis after a complete restriction with EcoRI. PMID:8851562

  12. Exercise upregulates salivary amylase in humans (Review).

    PubMed

    Koibuchi, Eri; Suzuki, Yoshio

    2014-04-01

    The secretion of salivary α-amylase is influenced by adrenergic regulation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis; thus, exercise affects the levels of salivary α-amylase. Granger et al published a review in 2007 that focused attention on salivary α-amylase. In addition, a portable system for monitoring salivary α-amylase activity was launched in Japan at the end of 2005. The correlation between exercise and salivary α-amylase has since been extensively investigated. The present review summarizes relevant studies published in the English and Japanese literature after 2006. A search of the PubMed and CiNii databases identified 54 articles, from which 15 original articles were selected. The findings described in these publications indicate that exercise consistently increases mean salivary α-amylase activities and concentrations, particularly at an intensity of >70% VO2max in healthy young individuals. Thus, these studies have confirmed that salivary α-amylase levels markedly increase in response to physical stress. Salivary α-amylase levels may therefore serve as an effective indicator in the non-invasive assessment of physical stress. PMID:24669232

  13. Elevated Gene Copy Number Does Not Always Explain Elevated Amylase Activities in Fishes.

    PubMed

    German, Donovan P; Foti, Dolly M; Heras, Joseph; Amerkhanian, Hooree; Lockwood, Brent L

    2016-01-01

    Amylase activity variation in the guts of several model organisms appears to be explained by amylase gene copy number variation. We tested the hypothesis that amylase gene copy number is always elevated in animals with high amylolytic activity. We therefore sequenced the amylase genes and examined amylase gene copy number in prickleback fishes (family Stichaeidae) with different diets including two species of convergently evolved herbivores with the elevated amylase activity phenotype. We found elevated amylase gene copy number (six haploid copies) with sequence variation among copies in one herbivore (Cebidichthys violaceus) and modest gene copy number (two to three haploid copies) with little sequence variation in the remaining taxa, which included herbivores, omnivores, and a carnivore. Few functional differences in amylase biochemistry were observed, and previous investigations showed similar digestibility among the convergently evolved herbivores with differing amylase genetics. Hence, the phenotype of elevated amylase activity can be achieved by different mechanisms (i.e., elevated expression of fewer genes, increased gene copy number, or expression of more efficient amylase proteins) with similar results. Phylogenetic and comparative genomic analyses of available fish amylase genes show mostly lineage-specific duplication events leading to gene copy number variation, although a whole-genome duplication event or chromosomal translocation may have produced multiple amylase copies in the Ostariophysi, again showing multiple routes to the same result. PMID:27327179

  14. Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

    PubMed

    Shiosaki, K; Takata, K; Omichi, K; Tomita, N; Horii, A; Ogawa, M; Matsubara, K

    1990-05-14

    A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes. PMID:2197187

  15. Characterization of the L. manihotivorans alpha-amylase gene.

    PubMed

    Morlon-Guyot, J; Mucciolo-Roux, F; Rodriguez Sanoja, R; Guyot, J P

    2001-07-01

    Primers and probes were established from the sequences of the alpha-amylase genes (amyA) of L. amylovorus CIP 102989 and of L. plantarum A6 (Giraud and Cuny 1997). They were successfully used for the detection of the amyA gene in L. manihotivorans strain LMG 18010T and a 2842 bp region, containing the entire gene (2706 bp) with its putative promoter has been sequenced. More than 98% nucleotide sequence identities was found with L. amylovorus and L. plantarum amyA genes. The deduced amino acid sequence shares more than 96% amino acid sequence identities with L. amylovorus and L. plantarum alpha-amylases, and also 65% and 59% identities with the alpha-amylases of B. subtilis and S. bovis, respectively. The 3' terminal part of L. manihotivorans LMG 18010T amyA gene contained four repeated sequences (SRU). The amyA genes of the three lactobacilli species differed mainly in the number of SRU and in the size of the flanking regions of the SRU. PMID:11697143

  16. Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis.

    PubMed

    Brooker, J D; McCarthy, J M

    1997-09-01

    Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular alpha-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15-20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium. PMID:9236293

  17. Purification of a novel α-amylase inhibitor from local Himalayan bean (Phaseolus vulgaris) seeds with activity towards bruchid pests and human salivary amylase.

    PubMed

    Gupta, Mridu; Sharma, Pratima; Nath, Amarjit K

    2014-07-01

    Six bean (Phaseolus vulgaris L.) cultivars of Himalayan region were analysed for α- amylase inhibitor activity. The α-amylase inhibitor from seeds of screened bean cultivar KR-9, showing maximum inhibitory activity was purified using ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-100) and ion exchange chromatography (DEAE-Sephadex). The inhibitor was purified to homogeneity as judged by native-PAGE with 14.22 fold purification and 71.66% recovery. Purified inhibitor consisted of three subunits of molecular weight 15,488, 18,620 and 26,302 daltons, respectively as determined by SDS-PAGE. It was found to be heat stable up to 30 °C-40 °C and had two pH optima of 5.0 and 6.9. Nature of inhibition was found to be of non-competitive type. The purified inhibitor was found to be effective against α-amylases extracted from larvae of Callosobruchus chinensis, Tribolium castaneum and gut enzyme of Spodoptera littoralis. Larvae of Tribolium castaneum fed on flour mixed with purified inhibitor for 5 days showed 100% larval mortality. Purified α-amylase inhibitor was also found to inhibit human salivary α-amylase, suggesting its potential in prevention and therapy of obesity and use as drug design targets for treatment of diabetes. The gene encoding the inhibitor may be used to develop transgenic plants resistant against insect pests. PMID:24966421

  18. Action of human pancreatic and salivary alpha-amylases on maltooligosaccharides: evaluation of kinetic parameters.

    PubMed

    Saito, N; Horiuchi, T; Yoshida, M; Imai, T

    1979-10-01

    The kinetic studies on the reactions of human pancreatic and salivary alpha-amylases with several maltooligosaccharides (maltotetraose, maltopentaose, maltohexaose, and maltoheptaose) were carried out. The susceptibility to hydrolysis with human pancreatic alpha-amylase decreased in the order of maltopentaose, maltohexaose, maltotetraose, and maltoheptaose, while with human salivary alpha-amylase maltopentaose was hydrolysed slightly slower than maltohexaose but fairly faster than maltotetraose or maltoheptaose from a viewpoint of the rates of reactions based on the amount of substrate changed. The relative rates of production of substrates, utilized in the coupled yeast alpha-glucosidase reaction, increased in the order of maltoheptaose, maltohexaose, maltotetraose, and maltopentaose with human pancreatic alpha-amylase, while with human salivary alpha-amylase in the order of maltoheptaose, maltotetraose, maltohexaose, and maltopentaose. Thus, maltopentaose was considered to be the best substrate over maltotetraose, maltohexaose or maltoheptaose for the alpha-glucosidase coupled method of alpha-amylase determination. PMID:385176

  19. A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

    PubMed Central

    Uozumi, N; Sakurai, K; Sasaki, T; Takekawa, S; Yamagata, H; Tsukagoshi, N; Udaka, S

    1989-01-01

    The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes. Images PMID:2464578

  20. Structural forms of the human amylase locus and their relationships to SNPs, haplotypes, and obesity

    PubMed Central

    Usher, Christina L; Handsaker, Robert E; Esko, Tõnu; Tuke, Marcus A; Weedon, Michael N; Hastie, Alex R; Cao, Han; Moon, Jennifer E; Kashin, Seva; Fuchsberger, Christian; Metspalu, Andres; Pato, Carlos N; Pato, Michele T; McCarthy, Mark I; Boehnke, Michael; Altshuler, David M; Frayling, Timothy M; Hirschhorn, Joel N; McCarroll, Steven A

    2016-01-01

    Hundreds of genes reside in structurally complex, poorly understood regions of the human genome1-3. One such region contains the three amylase genes (AMY2B, AMY2A, and AMY1) responsible for digesting starch into sugar. The copy number of AMY1 is reported to be the genome’s largest influence on obesity4, though genome-wide association studies for obesity have found this locus unremarkable. Using whole genome sequence analysis3,5, droplet digital PCR6, and genome mapping7, we identified eight common structural haplotypes of the amylase locus that suggest its mutational history. We found that AMY1 copy number in individuals’ genomes is generally even (rather than odd) and partially correlates to nearby SNPs, which do not associate with BMI. We measured amylase gene copy number in 1,000 obese or lean Estonians and in two other cohorts totaling ~3,500 individuals. We had 99% power to detect the lower bound of the reported effects on BMI4, yet found no association. PMID:26098870

  1. a-Amylase activity during pullulan production and a-Amylase gene analyses of Aureobasidium pullulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungus Aureobasidium pullulans is the source of commercially produced pullulan, a high molecular weight polysaccharide that is used in the manufacture of edible films. It has been proposed that alpha-amylase negatively affects the molecular weight of pullulan in late cultures. Based on a recen...

  2. Relationship among physiological quality, heterosis, and amylase gene expression in maize seeds.

    PubMed

    Oliveira, G E; Von Pinho, E V R; Andrade, T; Souza, J C; Caixeta, F; Ferreira, R A D C

    2015-01-01

    In this study, we analyzed heterosis, amylase enzyme gene expression, and the physiological quality of maize seeds with different genotypes and sizes, which were subjected to aging and not subjected to aging. We used seeds from 2 maize lines that differed with regard to physiological quality, the hybrid, and the reciprocal hybrid; they were classified into 2 sizes and were subjected to aging and not subjected to aging. Physiological quality was assessed by performing tests for germination, emergence, emergence speed index, and artificial aging. Expressions of the genes alpha amylase B73, alpha amylase (LOC542522), isoamylase mRNA clone 353244, and the endogenous controls ubiquitin and alcohol dehydrogenase in the seeds were studied using quantitative real-time-polymerase chain reaction. We observed heterosis for seed quality and for expression of amylase genes in the genotypes studied. We found no difference in seed quality between large and small seeds. PMID:26345793

  3. [Cloning the alpha-amylase gene of Streptococcus bovis and its expression in Bacillus subtilis cells].

    PubMed

    Iakorski, P; Kuntsova, M M; Loseva, E F; Khasanov, F K

    1991-06-01

    The gene coding for alpha-amylase from the ruminant bacterium Streptococcus bovis was cloned on the plasmid pMX39 in Bacillus subtilis cells. An alpha-amylase positive colony was isolated in the initial screening of 3900 colonies on the medium containing insoluble starch. The size of the insert was approximately 2.8 kb. The recombinant plasmid was stably maintained in Bacillus subtilis cells under the nonselective conditions. PMID:1944323

  4. RAmy2A; a novel alpha-amylase-encoding gene in rice.

    PubMed

    Huang, N; Reinl, S J; Rodriguez, R L

    1992-02-15

    The structure and expression of the alpha-amylase-encoding gene, RAmy2A, are described. This only representative of the Amy2 subfamily in rice differs from other cereal alpha-amylase-encoding genes in several respects. It contains the largest introns of all the cereal alpha-amylase-encoding genes examined to date. Moreover, the second of three introns in this gene contains a long inverted repeat sequence that can potentially form a large and stable stem-loop structure in the unspliced RNA transcript. Finally, RAmy2A is constitutively expressed at very low levels in germinated seeds, root, etiolated leaves, immature seeds and callus. This is in marked contrast to the Amy2 genes of wheat and barley which are highly expressed in the aleurone layer of the germinated seeds. PMID:1541400

  5. Differential expression of rice alpha-amylase genes during seedling development under anoxia.

    PubMed

    Hwang, Y S; Thomas, B R; Rodriguez, R L

    1999-08-01

    The unique capability of rice (Oryza sativa L.) seedlings to grow under anoxic conditions may result in part from their ability to express alpha-amylase and maintain the supply of sugar needed for energy metabolism. Previous studies have demonstrated that under aerobic conditions the Amy1 and Amy2 subfamily genes are regulated primarily by phytohormones while the Amy3 subfamily genes are induced during sugar starvation. The expression patterns for these alpha-amylase genes were considerably different in anoxic vs. aerobic rice seedlings. The level of total alpha-amylase mRNA under anoxic conditions was decreased in aleurone layers while it increased in the embryo. Anoxic conditions greatly diminished the expression of the Amy1A gene in aleurone. Conversely, expression of many Amy3 subfamily genes was up-regulated and prolonged in embryo tissues under anoxic conditions. PMID:10527416

  6. Sugar-inducible expression of a gene for beta-amylase in Arabidopsis thaliana.

    PubMed Central

    Mita, S; Suzuki-Fujii, K; Nakamura, K

    1995-01-01

    The levels of beta-amylase activity and of the mRNA for beta-amylase in rosette leaves of Arabidopsis thaliana (L.) Heynh. increased significantly, with the concomitant accumulation of starch, when whole plants or excised mature leaves were supplied with sucrose. A supply of glucose or fructose, but not of mannitol or sorbitol, to plants also induced the expression of the gene for beta-amylase, and the induction occurred not only in rosette leaves but also in roots, stems, and bracts. These results suggest that the gene for beta-amylase of Arabidopsis is subject to regulation by a carbohydrate metabolic signal, and expression of the gene in various tissues may be regulated by the carbon partitioning and sink-source interactions in the whole plant. The sugar-inducible expression of the gene in Arabidopsis was severely repressed in the absence of light. The sugar-inducible expression in the light was not inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea or by chloramphenicol, but it was inhibited by cycloheximide. These results suggest that a light-induced signal and de novo synthesis of proteins in the cytoplasm are involved in the regulation. A fusion gene composed of the 5' upstream region of the gene for beta-amylase from Arabidopsis and the coding sequence of beta-glucuronidase showed the sugar-inducible expression in a light-dependent manner in rosette leaves of transgenic Arabidopsis. PMID:7716246

  7. Amyrel, a paralogous gene of the amylase gene family in Drosophila melanogaster and the Sophophora subgenus

    PubMed Central

    Da Lage, Jean-Luc; Renard, Emmanuelle; Chartois, Frédérique; Lemeunier, Françoise; Cariou, Marie-Louise

    1998-01-01

    We describe a gene from Drosophila melanogaster related to the alpha-amylase gene Amy. This gene, which exists as a single copy, was named Amyrel. It is strikingly divergent from Amy because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes. Amyrel has also been cloned in Drosophila ananassae, Drosophila pseudoobscura, and Drosophila subobscura and is likely to be present throughout the Sophophora subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5′ and 3′ flanking regions between Amyrel genes from different species, which is not the case for Amy and which suggests that selection acts on these regions. In contrast to the Amy genes, Amyrel is transcribed in larvae of D. melanogaster but not in adults. However, the protein has not been detected yet. Amyrel evolves about twice as fast as Amy in the several species studied. We suggest that this gene could result from a duplication of Amy followed by accelerated and selected divergence toward a new adaptation. PMID:9618501

  8. Structure of amylase-binding protein A of Streptococcus gordonii: A potential receptor for human salivary α-amylase enzyme

    PubMed Central

    Sethi, Ashish; Mohanty, Biswaranjan; Ramasubbu, Narayanan; Gooley, Paul R

    2015-01-01

    Amylase-binding protein A (AbpA) of a number of oral streptococci is essential for the colonization of the dental pellicle. We have determined the solution structure of residues 24–195 of AbpA of Streptococcus gordonii and show a well-defined core of five helices in the region of 45–115 and 135–145. 13Cα/β chemical shift and heteronuclear 15N-{1H} NOE data are consistent with this fold and that the remainder of the protein is unstructured. The structure will inform future molecular experiments in defining the mechanism of human salivary α-amylase binding and biofilm formation by streptococci. PMID:25739638

  9. alpha. -Amylase of Clostridium thermosulfurogenes EM1: Nucleotide sequence of the gene, processing of the enzyme, and comparison to other. alpha. -amylases

    SciTech Connect

    Bahl, H.; Burchhardt, G.; Spreinat, A.; Haeckel, K.; Wienecke, A.; Antranikian, G.; Schmidt, B. )

    1991-05-01

    The nucleotide sequence of the {alpha}-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes Em1 suggested that the {alpha}-amylase is translated form mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature {alpha}-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 {alpha}-amylase with those from other bacterial and eukaryotic {alpha}-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca{sup 2+}-binding site (consensus region I) of this Ca{sub 2+}-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the {alpha}-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the {beta}-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.

  10. Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant constructs of barley '-amylase and Lentinula edodes glucoamylase genes were integrated into the chromosomes of Saccharomyces cerevisiae. The insertion was confirmed by PCR amplification of the gene sequence in the chromosomes. The expression was analyzed by SDS-PAGE of the enzymes puri...

  11. A lectin gene encodes the alpha-amylase inhibitor of the common bean.

    PubMed Central

    Moreno, J; Chrispeels, M J

    1989-01-01

    An alpha-amylase inhibitor that inhibits insect and mammalian alpha-amylases but not plant alpha-amylases, is present in seeds of the common bean (Phaseolus vulgaris). We have purified the alpha-amylase inhibitor by using a selective heat treatment in acidic medium and affinity chromatography with porcine pancreas alpha-amylase coupled to agarose. Under sodium dodecyl sulfate gel electrophoresis, the purified inhibitor gave rise to five bands with mobilities corresponding to molecular masses ranging from 14 to 19 kDa. N-terminal sequencing (up to 15 amino acids) of the polypeptides obtained from these bands resulted in only two different sequences matching two stretches of the amino acid sequence deduced from an already described lectin gene [Hoffman, L. M. (1984) J. Mol. Appl. Gen. 2,447-453]. This gene is different from but closely related to the genes that code for phytohemagglutinin, the major lectin of bean. Further evidence based on amino acid composition, identification of a precursor, and recognition of the product of the gene (expressed in Escherichia coli) by an anti-alpha-amylase inhibitor serum confirms that the inhibitor is encoded by this or a closely related lectin gene. This finding assigns a biological function, which has been described at the molecular level, to a plant lectin gene product and supports the defense role postulated for seed lectins. The lack of homology with other families of enzyme inhibitors suggests that this may be the first member of a new family of plant enzyme inhibitors. Images PMID:2682631

  12. Effect of anthranilic acid on the catabolite repression of a Drosophila amylase gene in E. coli

    SciTech Connect

    Stevens, S.M.; Moehring, J.M.; Chernin, M.I.

    1987-05-01

    A Drosophila pseudoobscura amylase pseudogene cloned in Escherichia coli is expressed at high levels. The expression of this pseudogene is repressed when glucose (0.5% final conc) is added to a starch minimal medium culture of E. coli cells that contain the amylase plasmid pAMY17F. Addition of anthranilic acid (7 mM final conc.) to catabolite repressed cells acts like adenosine 3',5' cyclic monophosphate (cAMP) by derepressing the amylase pseudogene at the promoter. This is consistent with the Metabolite Gene Regulation (MGR) model proposed by Kline et al. which suggests that small molecules can circumvent the necessity for cAMP. Catabolite repression of the amylase structural gene of D. pseudoobscura has been previously shown. This would suggest that the amylase pseudogene expression in E. coli is either from a Drosophila structural gene promoter co-cloned with the pseudogene or a catabolite repressible E. coli promoter placed in the proper orientation and reading frame during the rearrangement of pAMY17F.

  13. Prevalence of the Amylase-Binding Protein A Gene (abpA) in Oral Streptococci

    PubMed Central

    Brown, Alan E.; Rogers, Jeffrey D.; Haase, Elaine M.; Zelasko, Peter M.; Scannapieco, Frank A.

    1999-01-01

    Salivary amylase binds specifically to a number of oral streptococcal species. This interaction may play an important role in dental plaque formation. Recently, a 585-bp gene was cloned and sequenced from Streptococcus gordonii Challis encoding a 20.5-kDa amylase-binding protein (AbpA). The goal of this study was to determine if related genes are present in other species of oral streptococci. Biotinylated abpA was used in Southern blot analysis to screen genomic DNA from several strains representing eight species of oral streptococci. This probe hybridized with a 4.0-kb HindIII restriction fragment from all 13 strains of S. gordonii tested. The probe did not appear to bind to any restriction fragments from other species of amylase-binding oral streptococci including Streptococcus mitis (with the exception of 1 of 14 strains), Streptococcus crista (3 strains), Streptococcus anginosus (1 strain), and Streptococcus parasanguinis (1 strain), or to non-amylase-binding oral streptococci including Streptococcus sanguinis (3 strains), Streptococcus oralis (4 strains), and Streptococcus mutans (1 strain). Primers homologous to sequences within the 3′ and 5′ ends of abpA yielded products of 400 bp following PCR of genomic DNA from the Southern blot-positive strains. Several of these PCR products were cloned and sequenced. The levels of similarity of these cloned products to the abpA of S. gordonii Challis ranged from 91 to 96%. These studies reveal that the abpA gene appears to be specific to S. gordonii and differs from genes encoding amylase-binding proteins from other species of amylase-binding streptococci. PMID:10565935

  14. Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori.

    PubMed

    Korman, D R; Bayliss, F T; Barnett, C C; Carmona, C L; Kodama, K H; Royer, T J; Thompson, S A; Ward, M; Wilson, L J; Berka, R M

    1990-03-01

    Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional. PMID:2340591

  15. The bean. alpha. -amylase inhibitor is encoded by a lectin gene

    SciTech Connect

    Moreno, J.; Altabella, T.; Chrispeels, M.J. )

    1989-04-01

    The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has the same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.

  16. Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus alpha-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).

    PubMed

    Kim, Young-Hwa; Woloshuk, Charles P; Cho, Eun Hee; Bae, Jung Myung; Song, Young-Sun; Huh, Gyung Hye

    2007-04-01

    Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection. PMID:17149640

  17. Differential RNA Expression of Two Barley ß-Amylase Genes (Bmy1 and Bmy2) in Developing Grains and Their Association with ß-Amylase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA expression from the barley ß-amylase1 (Bmy1) gene was determined during seed development in four genotypes (Legacy, Harrington, Ashqelon, and PI 296897). The Bmy1 transcript amount in Legacy and Harrington was not significantly different at 17, 19, or 21 days after anthesis (DAA). Ashqelon Bmy...

  18. Effect of an herb root extract, herbal dentifrice and synthetic dentifrice on human salivary amylase

    PubMed Central

    Sapra, Gaurav; Vyas, Yogesh Kumar; Agarwal, Rahul; Aggarwal, Ashish; Chandrashekar, K T; Sharma, Kanika

    2013-01-01

    Background: Salivary amylase is an enzyme, which plays a vital role in formation of dental plaque. It has the ability to bind on the bacterial surfaces and to hydrolyze starch, giving rise to products that are transformed into acids leading to dental caries. Suppression of salivary amylase activity can lead to decrease in risk of dental caries and plaque associated periodontal diseases. The aim of this study was to evaluate the effect of an herb, Spilanthes calva (in form of a test dentifrice) on human salivary amylase activity and to compare it with other dentifrices. Materials and Methods: A total of 80 subjects of age 18-35 years were randomly selected and divided equally into 4 groups. Group 1 subjects were assigned to use Test Dentifrice (with S. calva root extract), while Group 2, Group 3, and Group 4 subjects were assigned to use Herbal Dentifrice (Arodent™), Synthetic Dentifrice (Colgate®), and Control Dentifrice respectively. Salivary amylase activity was determined by Bernfeld method in each group, before and after using the given dentifrices. Results: Maximum inhibition of salivary amylase activity was found in the group using test dentifrice as compared to others. Conclusion: The present study indicates that, the root extract of S. calva possess significant inhibitory activity for salivary amylase. Use of S. calva root extract will provide a wider protection against different pathogenic oral microflora. Use of this extract singly or in combination is strongly recommended in the dentifrice formulations. PMID:24130585

  19. Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli.

    PubMed

    Zafar, Asma; Aftab, Muhammad Nauman; ud Din, Zia; Aftab, Saima; Iqbal, Irfana; ul Haq, Ikram

    2016-02-01

    A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and β-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose. PMID:26526464

  20. The Amylase gene cluster on the evolving sex chromosomes of Drosophila miranda.

    PubMed

    Steinemann, S; Steinemann, M

    1999-01-01

    On the basis of chromosomal homology, the Amylase gene cluster in Drosophila miranda must be located on the secondary sex chromosome pair, neo-X (X2) and neo-Y, but is autosomally inherited in all other Drosophila species. Genetic evidence indicates no active amylase on the neo-Y chromosome and the X2-chromosomal locus already shows dosage compensation. Several lines of evidence strongly suggest that the Amy gene cluster has been lost already from the evolving neo-Y chromosome. This finding shows that a relatively new neo-Y chromosome can start to lose genes and hence gradually lose homology with the neo-X. The X2-chromosomal Amy1 is intact and Amy2 contains a complete coding sequence, but has a deletion in the 3'-flanking region. Amy3 is structurally eroded and hampered by missing regulatory motifs. Functional analysis of the X2-chromosomal Amy1 and Amy2 regions from D. miranda in transgenic D. melanogaster flies reveals ectopic AMY1 expression. AMY1 shows the same electrophoretic mobility as the single amylase band in D. miranda, while ectopic AMY2 expression is characterized by a different mobility. Therefore, only the Amy1 gene of the resident Amy cluster remains functional and hence Amy1 is the dosage compensated gene. PMID:9872956

  1. Cell-associated alpha-amylases of butyrate-producing Firmicute bacteria from the human colon.

    PubMed

    Ramsay, Alan G; Scott, Karen P; Martin, Jenny C; Rincon, Marco T; Flint, Harry J

    2006-11-01

    Selected butyrate-producing bacteria from the human colon that are related to Roseburia spp. and Butyrivibrio fibrisolvens showed a good ability to utilize a variety of starches for growth when compared with the Gram-negative amylolytic anaerobe Bacteroides thetaiotaomicron. A major cell-associated amylase of high molecular mass (140-210 kDa) was detected in each strain by SDS-PAGE zymogram analysis, and genes corresponding to these enzymes were analysed for two representative strains. Amy13B from But. fibrisolvens 16/4 is a multi-domain enzyme of 144.6 kDa that includes a family 13 glycoside hydrolase domain, and duplicated family 26 carbohydrate-binding modules. Amy13A (182.4 kDa), from Roseburia inulinivorans A2-194, also includes a family 13 domain, which is preceded by two repeat units of approximately 116 aa rich in aromatic residues, an isoamylase N-terminal domain, a pullulanase-associated domain, and an additional unidentified domain. Both Amy13A and Amy13B have N-terminal signal peptides and C-terminal cell-wall sorting signals, including a modified LPXTG motif similar to that involved in interactions with the cell surface in other Gram-positive bacteria, a hydrophobic transmembrane segment, and a basic C terminus. The overexpressed family 13 domains showed an absolute requirement for Mg2+ or Ca2+ for activity, and functioned as 1,4-alpha-glucanohydrolases (alpha-amylases; EC 3.2.1.1). These major starch-degrading enzymes thus appear to be anchored to the cell wall in this important group of human gut bacteria. PMID:17074899

  2. Disruption of amylase genes by RNA interference affects reproduction in the Pacific oyster Crassostrea gigas.

    PubMed

    Huvet, Arnaud; Béguel, Jean-Philippe; Cavaleiro, Nathalia Pereira; Thomas, Yoann; Quillien, Virgile; Boudry, Pierre; Alunno-Bruscia, Marianne; Fabioux, Caroline

    2015-06-01

    Feeding strategies and digestive capacities can have important implications for variation in energetic pathways associated with ecological and economically important traits, such as growth or reproduction in bivalve species. Here, we investigated the role of amylase in the digestive processes of Crassostrea gigas, using in vivo RNA interference. This approach also allowed us to investigate the relationship between energy intake by feeding and gametogenesis in oysters. Double-stranded (ds)RNA designed to target the two α-amylase genes A and B was injected in vivo into the visceral mass of oysters at two doses. These treatments caused significant reductions in mean mRNA levels of the amylase genes: -50.7% and -59% mRNA A, and -71.9% and -70.6% mRNA B in 15 and 75 µg dsRNA-injected oysters, respectively, relative to controls. Interestingly, reproductive knock-down phenotypes were observed for both sexes at 48 days post-injection, with a significant reduction of the gonad area (-22.5% relative to controls) and germ cell under-proliferation revealed by histology. In response to the higher dose of dsRNA, we also observed reductions in amylase activity (-53%) and absorption efficiency (-5%). Based on these data, dynamic energy budget modeling showed that the limitation of energy intake by feeding that was induced by injection of amylase dsRNA was insufficient to affect gonadic development at the level observed in the present study. This finding suggests that other driving mechanisms, such as endogenous hormonal modulation, might significantly change energy allocation to reproduction, and increase the maintenance rate in oysters in response to dsRNA injection. PMID:25883379

  3. Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity.

    PubMed

    Maurus, Robert; Begum, Anjuman; Williams, Leslie K; Fredriksen, Jason R; Zhang, Ran; Withers, Stephen G; Brayer, Gary D

    2008-03-18

    A mechanistic study of the essential allosteric activation of human pancreatic alpha-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic alpha-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic alpha-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic alpha-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic alpha-amylase and leads to the production of the same acarbose product

  4. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

    PubMed Central

    Satoh, E; Niimura, Y; Uchimura, T; Kozaki, M; Komagata, K

    1993-01-01

    The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148. Images PMID:8285674

  5. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

    PubMed

    Satoh, E; Niimura, Y; Uchimura, T; Kozaki, M; Komagata, K

    1993-11-01

    The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148. PMID:8285674

  6. Potent Human α-Amylase Inhibition by the β-Defensin-like Protein Helianthamide

    PubMed Central

    2016-01-01

    Selective inhibitors of human pancreatic α-amylase (HPA) are an effective means of controlling blood sugar levels in the management of diabetes. A high-throughput screen of marine natural product extracts led to the identification of a potent (Ki = 10 pM) peptidic HPA inhibitor, helianthamide, from the Caribbean sea anemone Stichodactyla helianthus. Active helianthamide was produced in Escherichia coli via secretion as a barnase fusion protein. X-ray crystallographic analysis of the complex of helianthamide with porcine pancreatic α-amylase revealed that helianthamide adopts a β-defensin fold and binds into and across the amylase active site, utilizing a contiguous YIYH inhibitory motif. Helianthamide represents the first of a novel class of glycosidase inhibitors and provides an unusual example of functional malleability of the β-defensin fold, which is rarely seen outside of its traditional role in antimicrobial peptides. PMID:27066537

  7. Cloning and Characterization of an Alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular a-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  8. Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Pusey, Mark L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular alpha-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  9. Expression of a bacterial alpha-amylase gene in transgenic rice seeds.

    PubMed

    Xu, Xiaoli; Fang, Jun; Wang, Wei; Guo, Jianli; Chen, Pinnan; Cheng, Jiaan; Shen, Zhicheng

    2008-08-01

    An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction. PMID:17926139

  10. Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome.

    PubMed Central

    Scheirlinck, T; Mahillon, J; Joos, H; Dhaese, P; Michiels, F

    1989-01-01

    A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria. Images PMID:2679379

  11. Metabolism of glycosylated human salivary amylase: in vivo plasma clearance by rat hepatic endothelial cells and in vitro receptor mediated pinocytosis by rat macrophages

    SciTech Connect

    Niesen, T.E.; Alpers, D.H.; Stahl, P.D.; Rosenblum, J.L.

    1984-09-01

    Salivary-type amylase normally comprises about 60% of the amylase activity in human serum, but only a small fraction is a glycosylated isoenzyme (amylase A). In contrast, 1/3 of amylase in human saliva is glycosylated. Since glycosylation can affect circulatory clearance, we studied the clearance of amylase A in rats and its uptake by rat alveolar macrophages. Following intravenous injection, /sup 125/I-labeled amylase A disappeared rapidly from plasma (t 1/2 . 9 min) and accumulated in the liver. Simultaneous injection of mannose-albumin slowed its clearance to a rate comparable to that of /sup 125/I-labeled nonglycosylated salivary amylase (t 1/2 . 45 min). In contrast, galactose-albumin had no effect. Electron microscope autoradiography of the liver following injection of /sup 125/I-labeled amylase A revealed a localization of grains over the hepatic endothelial cells. In vitro studies indicated that amylase A is taken up by alveolar macrophages via receptor-mediated pinocytosis. Uptake was linear over time, saturable, and inhibited by mannan and mannose-albumin, but not by galactose-albumin. We conclude that amylase A, which is a naturally occurring human glycoprotein with at most three terminal L-fucose residues per molecule, is recognized in rats by a mannose receptor located on hepatic endothelial cells. We speculate that this receptor, by rapidly clearing circulating amylase A, may be responsible for the low level of amylase A in human serum.

  12. Low copy number of the salivary amylase gene predisposes to obesity.

    PubMed

    Falchi, Mario; El-Sayed Moustafa, Julia Sarah; Takousis, Petros; Pesce, Francesco; Bonnefond, Amélie; Andersson-Assarsson, Johanna C; Sudmant, Peter H; Dorajoo, Rajkumar; Al-Shafai, Mashael Nedham; Bottolo, Leonardo; Ozdemir, Erdal; So, Hon-Cheong; Davies, Robert W; Patrice, Alexandre; Dent, Robert; Mangino, Massimo; Hysi, Pirro G; Dechaume, Aurélie; Huyvaert, Marlène; Skinner, Jane; Pigeyre, Marie; Caiazzo, Robert; Raverdy, Violeta; Vaillant, Emmanuel; Field, Sarah; Balkau, Beverley; Marre, Michel; Visvikis-Siest, Sophie; Weill, Jacques; Poulain-Godefroy, Odile; Jacobson, Peter; Sjostrom, Lars; Hammond, Christopher J; Deloukas, Panos; Sham, Pak Chung; McPherson, Ruth; Lee, Jeannette; Tai, E Shyong; Sladek, Robert; Carlsson, Lena M S; Walley, Andrew; Eichler, Evan E; Pattou, Francois; Spector, Timothy D; Froguel, Philippe

    2014-05-01

    Common multi-allelic copy number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts. Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene (AMY1) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 copy number was positively associated with both amylase gene expression (P = 2.31 × 10(-14)) and serum enzyme levels (P < 2.20 × 10(-16)), whereas reduced AMY1 copy number was associated with increased BMI (change in BMI per estimated copy = -0.15 (0.02) kg/m(2); P = 6.93 × 10(-10)) and obesity risk (odds ratio (OR) per estimated copy = 1.19, 95% confidence interval (CI) = 1.13-1.26; P = 1.46 × 10(-10)). The OR value of 1.19 per copy of AMY1 translates into about an eightfold difference in risk of obesity between subjects in the top (copy number > 9) and bottom (copy number < 4) 10% of the copy number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies. PMID:24686848

  13. Crystal structures of human pancreatic alpha-amylase in complex with carbohydrate and proteinaceous inhibitors.

    PubMed Central

    Nahoum, V; Roux, G; Anton, V; Rougé, P; Puigserver, A; Bischoff, H; Henrissat, B; Payan, F

    2000-01-01

    Crystal structures of human pancreatic alpha-amylase (HPA) in complex with naturally occurring inhibitors have been solved. The tetrasaccharide acarbose and a pseudo-pentasaccharide of the trestatin family produced identical continuous electron densities corresponding to a pentasaccharide species, spanning the -3 to +2 subsites of the enzyme, presumably resulting from transglycosylation. Binding of the acarviosine core linked to a glucose residue at subsites -1 to +2 appears to be a critical part of the interaction process between alpha-amylases and trestatin-derived inhibitors. Two crystal forms, obtained at different values of pH, for the complex of HPA with the protein inhibitor from Phaseolus vulgaris (alpha-amylase inhibitor) have been solved. The flexible loop typical of the mammalian alpha-amylases was shown to exist in two different conformations, suggesting that loop closure is pH-sensitive. Structural information is provided for the important inhibitor residue, Arg-74, which has not been observed previously in structural analyses. PMID:10657258

  14. Interaction mechanism between green tea extract and human α-amylase for reducing starch digestion.

    PubMed

    Miao, Ming; Jiang, Bo; Jiang, Huan; Zhang, Tao; Li, Xingfeng

    2015-11-01

    This study evaluated the inhibitory effects of the green tea extract on human pancreatic α-amylase activity and its molecular mechanism. The green tea extract was composed of epicatechin (59.2%), epigallocatechin gallate (14.6%) and epicatechin gallate (26.2%) as determined by HPLC analysis. Enzyme activity measurement showed that % inhibition and IC50 of the green tea extract (10%, based on starch) were 63.5% and 2.07 mg/ml, respectively. The Michaelis-Menten constant remained unchanged but the maximal velocity decreased from 0.43 (control) to 0.07 mg/(ml × min) (4 mg/ml of the green tea extract), indicating that the green tea extract was an effective inhibitor against α-amylase with a non-competitive mode. The fluorescence data revealed that the green tea extract bound with α-amylase to form a new complex with static quenching mechanism. Docking study showed the epicatechin gallate in the green tea extract presented stronger affinity than epigallocatechin gallate, with more number of amino acid residues involved in amylase binding with hydrogen bonds and Van der Waals forces. Thus, the green tea extract could be used to manipulate starch digestion for potential health benefits. PMID:25976786

  15. Cloning, enhanced expression and characterization of an α-amylase gene from a wild strain in B. subtilis WB800.

    PubMed

    Chen, Jing; Chen, Xianghua; Dai, Jun; Xie, Guangrong; Yan, Luying; Lu, Lina; Chen, Jianhua

    2015-09-01

    A Bacillus strain with high productivity of α-amylase isolated from a starch farm was identified as Bacillus amyloliquefaciens. The α-amylase encoding gene amy1 was cloned into pMD18-T vector and amplified in E. coli DH5α. Shuttle vector pP43MNX was reconstructed to obtain vector pP43X for heterologous expression of the α-amylase in B. subtilis WB800. Recombinant enzyme was sufficiently purified by precipitation, gel filtration and anion exchange with a specific activity of 5566 U/mg. The α-amylase sequence contains an open reading frame of 1545 bp, which encodes a protein of 514 amino acid residues with a predicted molecular mass of 58.4 kDa. The enzyme exhibited maximal activity at pH 6.0 and 60 °C. Catalytic efficiency of the recombinant α-amylase was inhibited by Hg(2+), Pb(2+) and Cu(2+), but stimulated by Li(+), Mn(2+) and Ca(2+). The purified enzyme showed decreased activity toward detergents (SDS, Tween 20 and Triton X-100). Compared with production by the wild strain, there was a 1.48-fold increase in the productivity of α-amylase in recombinant B. subtilis WB800. PMID:26092061

  16. Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura.

    PubMed

    Popadić, A; Anderson, W W

    1995-07-01

    The alpha-amylase (Amy) multigene family in Drosophila pseudoobscura is located on the third chromosome, which is polymorphic for more than 40 inverted gene arrangements. The number of copies in this family ranges from one to three, depending on the arrangement in question. A previous study of the three Amy genes from the Standard (ST) arrangement suggested either that duplicated copies (Amy2 and Amy3) are functionally constrained or that they are undergoing gene conversion with Amy1. In order to elucidate further the pattern of molecular evolution in this family, we cloned and sequenced four additional Amy genes, two from the Santa Cruz (SC) and two from the Chiricahua (CH) gene arrangement. Of the two alternatives, only the hypothesis of gene conversion is supported by the sequence analysis. The homogenization effect of gene conversion has been strongest in SC, whose copies differ by only two nucleotides, less noticeable in ST, and negligible in the CH. Furthermore, the action of gene conversion is apparently localized, occurring only in the coding region. Interestingly, these results concur with the findings of other workers for the duplicated Amy genes in the Drosophila melanogaster group. Thus, the occurrence of gene conversion in the Amy multigene family seems to be a common feature in the Drosophila species studied so far. PMID:7659012

  17. Three alpha-amylase genes of Aspergillus oryzae exhibit identical intron-exon organization.

    PubMed

    Wirsel, S; Lachmund, A; Wildhardt, G; Ruttkowski, E

    1989-01-01

    We have cloned three genes (amy1, amy2 and amy3) encoding alpha-amylase in the filamentous fungus Aspergillus oryzae. The established overall sequences have a very high degree of homology, showing divergences mainly in the 3'-untranslated regions. The positions and the sequences of the eight introns were found to be absolutely identical in the three genes. The sequence analysis of the 5'-regions revealed presumptive TATA, CAAT and GC boxes. Primer extension analysis was performed to determine the transcription start. We were able to detect mRNAs from amy1 and amy3 but not from amy2 with gene-specific oligonucleotide probes complementary to the 3'-noncoding regions. PMID:2785629

  18. Molecular evolution of dimeric α-amylase inhibitor genes in wild emmer wheat and its ecological association

    PubMed Central

    2008-01-01

    Background α-Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. In this study, we aimed to reveal the structure and diversity of dimeric α-amylase inhibitor genes in wild emmer wheat from Israel and to elucidate the relationship between the emmer wheat genes and ecological factors using single nucleotide polymorphism (SNP) markers. Another objective of this study was to find out whether there were any correlations between SNPs in functional protein-coding genes and the environment. Results The influence of ecological factors on the genetic structure of dimeric α-amylase inhibitor genes was evaluated by specific SNP markers. A total of 244 dimeric α-amylase inhibitor genes were obtained from 13 accessions in 10 populations. Seventy-five polymorphic positions and 74 haplotypes were defined by sequence analysis. Sixteen out of the 75 SNP markers were designed to detect SNP variations in wild emmer wheat accessions from different populations in Israel. The proportion of polymorphic loci P (5%), the expected heterozygosity He, and Shannon's information index in the 16 populations were 0.887, 0.404, and 0.589, respectively. The populations of wild emmer wheat showed great diversity in gene loci both between and within populations. Based on the SNP marker data, the genetic distance of pair-wise comparisons of the 16 populations displayed a sharp genetic differentiation over long geographic distances. The values of P, He, and Shannon's information index were negatively correlated with three climatic moisture factors, whereas the same values were positively correlated by Spearman rank correlation coefficients' analysis with some of the other ecological factors. Conclusion The populations of wild emmer wheat showed a wide range of diversity in dimeric α-amylase inhibitors, both between and within populations. We suggested that SNP markers are useful for the estimation of

  19. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat Butte 86

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complement of genes encoding alpha-amylase/protease inhibitors expressed in Triticum aestivum cv. Butte 86 was characterized by transcript and proteomic analysis. Coding sequences for 18 distinct proteins were identified among a collection of expressed sequence tags (ESTs) from Butte 86 developi...

  20. Codon Optimization Significantly Improves the Expression Level of α -Amylase Gene from Bacillus licheniformis in Pichia pastoris.

    PubMed

    Wang, Jian-Rong; Li, Yang-Yuan; Liu, Dan-Ni; Liu, Jing-Shan; Li, Peng; Chen, Li-Zhi; Xu, Shu-De

    2015-01-01

    α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use. PMID:26171389

  1. Heterotrimeric G proteins are implicated in gibberellin induction of a-amylase gene expression in wild oat aleurone

    PubMed

    Jones; Smith; Desikan; Plakidou-Dymock; Lovegrove; Hooley

    1998-02-01

    The role of heterotrimeric G proteins in gibberellin (GA) induction of a-amylase gene expression was examined in wild oat aleurone protoplasts. Mas7, a cationic amphiphilic tetradecapeptide that stimulates GDP/GTP exchange by heterotrimeric G proteins, specifically induced alpha-amylase gene expression and enzyme secretion in a very similar manner to GA1. In addition, Mas7 stimulated expression of an alpha-Amy2/54:GUS promoter and reporter construct in transformed protoplasts. Both Mas7 and GA1 induction of alpha-amylase mRNA were insensitive to pertussis toxin. Hydrolysis-resistant nucleotides were introduced into aleurone protoplasts during transfection with reporter gene constructs. GDP-beta-S, which inhibits GDP/GTP exchange by heterotrimeric G proteins, completely prevented GA1 induction of alpha-Amy2/54:GUS expression, whereas GTP-gamma-S, which activates heterotrimeric G proteins, stimulated expression very slightly. Novel cDNA sequences from Galpha and Gbeta subunits were cloned from wild oat aleurone cells. By using RNA gel blot analysis, we found that the transcripts were expressed at a low level. Heterotrimeric G proteins have been implicated in several events during plant growth and development, and these data suggest that they may be involved in GA regulation of alpha-amylase gene expression in aleurone. PMID:9490747

  2. Codon Optimization Significantly Improves the Expression Level of α-Amylase Gene from Bacillus licheniformis in Pichia pastoris

    PubMed Central

    Wang, Jian-Rong; Li, Yang-Yuan; Liu, Dan-Ni; Liu, Jing-Shan; Li, Peng; Chen, Li-Zhi; Xu, Shu-De

    2015-01-01

    α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use. PMID:26171389

  3. Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger.

    PubMed

    Juge, N; Svensson, B; Williamson, G

    1998-04-01

    Efficient production of recombinant barley alpha-amylase has been achieved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44,960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley alpha-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence. PMID:9615479

  4. Variation in salivary and pancreatic alpha-amylase genes in Italian horse breeds.

    PubMed

    Coizet, Beatrice; Nicoloso, Letizia; Marletta, Donata; Tamiozzo-Calligarich, Alessandra; Pagnacco, Giulio; Crepaldi, Paola

    2014-01-01

    The dietary demand of the modern horse relies on high-cereal feeding and limited forage compared with natural grazing conditions, predisposing the horse to several important diseases. Salivary and pancreatic alpha-amylases (coded by AMY1 and AMY2 genes, respectively) play a crucial role in carbohydrate digestion in nonruminants, but little is known about these 2 genes in the horse. Aim of this work has been to distinguish genomic sequences of horse AMY1 and AMY2 genes and to analyze any polymorphisms in breeds historically characterized by marked differences in nutritional management. A single nucleotide polymorphism detection was performed and 7 novel single nucleotide polymorphisms were found. Three single nucleotide polymorphisms are in exons and were genotyped in 112 horses belonging to 6 breeds. One single nucleotide polymorphism in AMY1 gene distinguished Haflinger and the Italian native Murgese from the other breeds, whereas both the single nucleotide polymorphisms in AMY2 gene showed different allelic frequencies in Friesian compared with the other breeds. These differences are confirmed by quite high fixation index (Fst) values for these 2 nonsynonymous single nucleotide polymorphisms. These preliminary results highlight marked divergences in allele frequencies of AMY1 and AMY2 genes, involved in starch digestion, between horse breeds characterized by different histories of selection, thus providing first indications of possible relations between genetics and nutritional management. PMID:24558100

  5. Amylase mRNA transcripts in normal tissues and neoplasms: the implication of different expressions of amylase isogenes.

    PubMed

    Seyama, K; Nukiwa, T; Takahashi, K; Takahashi, H; Kira, S

    1994-01-01

    To understand the cellular origin and mechanism of gene expression in amylase-producing cancers, the phenotyping of amylase isogenes by the polymerase chain reaction and restriction-fragment-length polymorphism using restriction endonucleases TaqI, DdeI, HinfI, and AfaI were performed for 3 amylase-producing lung adenocarcinomas, 16 lung cancers without hyperamylasemia, other human malignant neoplasms, cultured cell lines, and normal tissues. In addition, amylase mRNA transcripts were semi-quantified by the limited polymerase chain reaction. Amylase mRNA transcripts were detected in all of the tissues examined. The AMY1 gene (salivary type) was exclusively and highly expressed in the salivary glands and the amylase-producing lung adenocarcinomas. Coexpression of the AMY1 gene and AMY2 gene (pancreatic type) was observed in most of the lung cancers without hyperamylasemia, lung tissue, and cells scraped from the tracheal epithelium, thyroid, and female genital tract (ovary, fallopian tube, and uterus cervix), while minimal levels of mRNA transcripts of the AMY2 gene were detected in other malignant neoplasms, various normal tissues, and the cultured cell lines. All mRNA transcripts identified as being those of the AMY2 gene were further identified as being from the AMY2B gene except for the transcripts from the pancreas, in which the AMY2A gene and AMY2B gene were coexpressed. On the basis of these results, the clinical occurrence of amylase-producing cancer likely relates to the tissues expressing the AMY1 gene, while the AMY2B gene, which evolutionarily is the oldest gene among human amylase isogenes, is constitutively expressed in various tissues. PMID:7507116

  6. Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated alpha-amylase genes.

    PubMed

    Rushton, P J; Hooley, R; Lazarus, C M

    1992-09-01

    Binding of nuclear proteins from wild oat aleurone protoplasts to the promoter regions of two gibberellin-regulated wheat alpha-amylase genes (alpha-Amy1/18 and alpha-Amy2/54) has been studied by gel retardation and DNase 1 footprinting. Gel retardation studies using 300-430 bp fragments of the promoters showed similar binding characteristics with nuclear extracts from both gibberellin A1-treated and untreated protoplasts. DNase 1 footprints localised binding of nuclear proteins from gibberellin A1-treated aleurone protoplasts to regions in both promoters. Similar sequence elements in the promoter regions of both genes were protected from digestion although the location and number of footprints in each promoter region were different. Each footprint contained either a sequence similar to the cAMP and/or phorbol ester response elements, or a hyphenated palindrome sequence. The presence of cAMP and/or phorbol ester response element-like sequences in the footprints suggests that transcription factors of the bZIP type may be involved in the expression of alpha-amylase genes in aleurone cells. Footprints containing hyphenated palindrome sequences, found in the promoter regions of both genes, suggest the possible involvement of other classes of transcription factor. The conserved alpha-amylase promoter sequence TAA-CAGA was also shown to bind nuclear protein in the alpha-Amy2/54 promoter. These observations are discussed in relation to alpha-amylase gene expression in aleurone and to functional data concerning these genes. PMID:1511135

  7. Tobacco Plants Transformed with the Bean αai Gene Express an Inhibitor of Insect α-Amylase in Their Seeds 1

    PubMed Central

    Altabella, Teresa; Chrispeels, Maarten J.

    1990-01-01

    Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant α-amylases. We recently (J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889) presented strong circumstantial evidence that this α-amylase inhibitor (αAI) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5′ and 3′ flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (Mr 10,000-18,000) that cross-react with antibodies to the bean α-amylase inhibitor. Most of these polypeptides bind to a pig pancreas α-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas α-amylase activity as well as the α-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called αai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera. Images Figure 2 Figure 3 Figure 5 PMID:16667540

  8. Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

    SciTech Connect

    Altabella, T.; Chrispeels, M.J. )

    1990-06-01

    Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{sub r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.

  9. Stability of human α-salivary amylase in aged forensic samples.

    PubMed

    Carboni, Ilaria; Rapi, Stefano; Ricci, Ugo

    2014-07-01

    The unequivocal tissue identification in forensic casework samples is a key step for crime scene reconstruction. Just knowing the origin of a fluid can sometimes be enough to either prove or disprove a fact in court. Despite the importance of this test, very few data are available in literature concerning human saliva identification in old forensic caseworks. In this work the stability of human α-amylase activity in aged samples is described by using three different methods integrated with DNA profiling techniques. This analytical protocol was successfully applied on 26-years old samples coming from anonymous threat letters sent to prosecutors who were working on "the Monster of Florence", a case of serial murders happened around Florence (Italy) between 1968 and 1985. PMID:24755314

  10. Amylase - blood

    MedlinePlus

    ... amylase levels may occur due to: Acute pancreatitis Cancer of the pancreas , ovaries, or lungs Cholecystitis Gallbladder attack caused by ... open) Decreased amylase levels may occur due to: Cancer of the pancreas Damage to the pancreas Kidney disease Toxemia of ...

  11. Lactase persistence and augmented salivary alpha-amylase gene copy numbers might have been selected by the combined toxic effects of gluten and (food born) pathogens.

    PubMed

    Pruimboom, Leo; Fox, Tom; Muskiet, Frits A J

    2014-03-01

    Various positively selected adaptations to new nutrients have been identified. Lactase persistence is among the best known, conferring the ability for drinking milk at post weaning age. An augmented number of amylase gene (AMY1) copies, giving rise to higher salivary amylase activity, has been implicated in the consumption of starch-rich foods. Higher AMY1 copy numbers have been demonstrated in populations with recent histories of starchy-rich diets. It is however questionable whether the resulting polymorphisms have exerted positive selection only by providing easily available sources of macro and micronutrients. Humans have explored new environments more than any other animal. Novel environments challenge the host, but especially its immune system with new climatic conditions, food and especially pathogens. With the advent of the agricultural revolution and the concurrent domestication of cattle came new pathogens. We contend that specific new food ingredients (e.g., gluten) and novel pathogens drove selection for lactase persistence and higher AMY gene copy numbers. Both adaptations provide ample glucose for activating the sodium glucose-dependent co-transporter 1 (SGLT1), which is the principal glucose, sodium and water transporter in the gastro-intestinal tract. Their rapid uptake confers protection against potentially lethal dehydration, hyponatremia and ultimately multiple organ failure. Oral rehydration therapy aims at SGLT1 activity and is the current treatment of choice for chronic diarrhoea and vomiting. We hypothesize that lifelong lactase activity and rapid starch digestion should be looked at as the evolutionary covalent of oral rehydration therapy. PMID:24472865

  12. Salivary α-amylase exhibits antiproliferative effects in primary cell cultures of rat mammary epithelial cells and human breast cancer cells

    PubMed Central

    2011-01-01

    Background Breast cancer is one of the most diagnosed cancers in females, frequently with fatal outcome, so that new strategies for modulating cell proliferation in the mammary tissue are urgently needed. There is some, as yet inconclusive evidence that α-amylase may constitute a novel candidate for affecting cellular growth. Methods The present investigation aimed to examine if salivary α-amylase, an enzyme well known for the metabolism of starch and recently introduced as a stress marker, is able to exert antiproliferative effects on the growth of mammary gland epithelial cells. For this purpose, primary epithelial cultures of breast tissue from two different inbred rat strains, Fischer 344 (F344) and Lewis, as well as breast tumor cells of human origin were used. Treatment with human salivary α-amylase was performed once daily for 2 days followed by cell counting (trypan blue assay) to determine alterations in cell numbers. Cell senescence after α-amylase treatment was assessed by β-galactosidase assay. Endogenous α-amylase was detected in cells from F344 and Lewis by immunofluorescence. Results Salivary α-amylase treatment in vitro significantly decreased the proliferation of primary cells from F344 and Lewis rats in a concentration-dependent manner. Noticeably, the sensitivity towards α-amylase was significantly higher in Lewis cells with stronger impact on cell growth after 5 and 50 U/ml compared to F344 cells. An antiproliferative effect of α-amylase was also determined in mammary tumor cells of human origin, but this effect varied depending on the donor, age, and type of the cells. Conclusions The results presented here indicate for the first time that salivary α-amylase affects cell growth in rat mammary epithelial cells and in breast tumor cells of human origin. Thus, α-amylase may be considered a novel, promising target for balancing cellular growth, which may provide an interesting tool for tumor prophylaxis and treatment. PMID:22027017

  13. Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents

    PubMed Central

    Zinjarde, Smita; Thulasiram, Hirekodathakallu; RaviKumar, Ameeta

    2015-01-01

    Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (Ki 42.2, 18.6 μM) and starch (Ki′ 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia. PMID:26469405

  14. Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents.

    PubMed

    Ponnusamy, Sudha; Haldar, Saikat; Mulani, Fayaj; Zinjarde, Smita; Thulasiram, Hirekodathakallu; RaviKumar, Ameeta

    2015-01-01

    Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (Ki 42.2, 18.6 μM) and starch (Ki' 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia. PMID:26469405

  15. Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae.

    PubMed

    Eksteen, Jeremy M; Steyn, Andries J C; van Rensburg, Pierre; Cordero Otero, Ricardo R; Pretorius, Isak S

    2003-01-15

    Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases

  16. Application of the extracellular alpha-amylase gene from Streptococcus bovis 148 to construction of a secretion vector for yogurt starter strains.

    PubMed

    Satoh, E; Ito, Y; Sasaki, Y; Sasaki, T

    1997-11-01

    Streptococcus thermophilus ATCC 19258, Lactobacillus delbrueckii subsp. bulgaricus T-11, and Lactococcus lactis subsp. lactis IL1403 were transformed with the alpha-amylase gene (amyA) from Streptococcus bovis 148 by using a wide host-range vector, and all the transformants secreted the alpha-amylase successfully. Since the promoter and the secretion signal of the amyA gene were functional in these strains, we constructed a secretion vector using the expression elements of amyA. Trials to secrete foreign enzymes in yogurt starter strains were performed using this novel secretion vector. PMID:9361445

  17. Application of the extracellular alpha-amylase gene from Streptococcus bovis 148 to construction of a secretion vector for yogurt starter strains.

    PubMed Central

    Satoh, E; Ito, Y; Sasaki, Y; Sasaki, T

    1997-01-01

    Streptococcus thermophilus ATCC 19258, Lactobacillus delbrueckii subsp. bulgaricus T-11, and Lactococcus lactis subsp. lactis IL1403 were transformed with the alpha-amylase gene (amyA) from Streptococcus bovis 148 by using a wide host-range vector, and all the transformants secreted the alpha-amylase successfully. Since the promoter and the secretion signal of the amyA gene were functional in these strains, we constructed a secretion vector using the expression elements of amyA. Trials to secrete foreign enzymes in yogurt starter strains were performed using this novel secretion vector. PMID:9361445

  18. Establishing the catalytic mechanism of human pancreatic α-amylase with QM/MM methods.

    PubMed

    Pinto, Gaspar P; Brás, Natércia F; Perez, Marta A S; Fernandes, Pedro A; Russo, Nino; Ramos, Maria J; Toscano, Marirosa

    2015-06-01

    In this work, we studied the catalytic mechanism of human pancreatic α-amylase (HPA). Our goal was to determine the catalytic mechanism of HPA with atomic detail using computational methods. We demonstrated that the HPA catalytic mechanism consists of two steps, the first of which (glycosylation step) involves breaking the glycosidic bond to culminate in the formation of a covalent intermediate. The second (deglycosylation step) consists of the addition of a water molecule to release the enzyme/substrate covalent intermediate, completing the hydrolysis of the sugar. The active site was very open to the solvent. Our mechanism basically differs from the previously proposed mechanism by having two water molecules instead of only one near the active site that participate in the mechanism. We also demonstrate the relevant role of the three catalytic amino acids, two aspartate residues and a glutamate (D197, E233, and D300), during catalysis. It was also shown that the rate limiting step was glycosylation, and its activation energy was in agreement with experimental values obtained for HPA. The experimental activation energy was 14.4 kcal mol(-1), and the activation energy obtained computationally was 15.1 kcal mol(-1). PMID:26575550

  19. Evolution of nucleotide substitutions and gene regulation in the amylase multigenes in Drosophila kikkawai and its sibling species.

    PubMed

    Inomata, N; Yamazaki, T

    2000-04-01

    In order to determine evolutionary changes in gene regulation and the nucleotide substitution pattern in a multigene family, the amylase multigenes were characterized in Drosophila kikkawai and its sibling species. The nucleotide substitution pattern was investigated. Drosophila kikkawai has four amylase genes. The Amy1 and Amy2 genes are a head-to-head duplication in the middle of the B arm of the second chromosome, while the Amy3 and Amy4 genes are a tail-to-tail duplication near the centromere of the same chromosome. In the sibling species of D. kikkawai (Drosophila bocki, Drosophila leontia, and Drosophila lini), sequencing of the Amy1, Amy2, Amy3, and Amy4 genes revealed that the Amy1 and Amy2 gene group diverged from Amy3 and Amy4 after duplication. In the Amy1 and Amy2 genes, the divergent evolution occurred in the flanking regions; in contrast, the coding regions have evolved in concerted fashion. The electrophoretic pattern of AMY isozymes was also examined. In D. kikkawai and its siblings, two or three electrophoretically different isozymes are encoded by the Amy1 and Amy2 genes (S isozyme) and by the Amy3 and Amy4 genes (F (M) isozymes). The S and F (M) isozymes show different patterns of band intensity when larvae and flies were fed in different media. Amy1 and Amy2, which encode the S isozyme, are more strikingly regulated than Amy3 and Amy4, which encode the F (M) isozyme. The GC content and codon usage bias were higher for the Amy1 and Amy2 genes than for the Amy3 and Amy4 genes. Although the ratio of synonymous and replacement substitutions within the Amy1 and Amy2 gene group was not significantly different from that within the Amy3 and Amy4 gene group, the synonymous substitution rate in the lineage of Amy1 and Amy2 was lower than that of Amy3 and Amy4. In conclusion, after the first duplication but before speciation of four species, the synonymous substitution rate between the two lineages and the electrophoretic pattern of the isozymes encoded by

  20. Negative regulation in the expression of a sugar-inducible gene in Arabidopsis thaliana. A recessive mutation causing enhanced expression of a gene for beta-amylase.

    PubMed Central

    Mita, S; Hirano, H; Nakamura, K

    1997-01-01

    Expression of a beta-amylase gene of Arabidopsis thaliana (AT beta-Amy) is regulated by sugars. We identified a mutant, hba1, in which the level of expression of AT beta-Amy in leaves of plants that had been grown in a medium with 2% sucrose was significantly higher than that in wild-type plants. Higher that wild-type levels of beta-amylase in hba1 plants depended on the presence of 1 to 2% sucrose or 1% glucose in the medium, whereas leaves of mutant plants grown with higher levels of sugars had beta-amylase activities similar to those in leaves of wild-type plants. The hba1 phenotype was recessive and did not affect levels of sugars and starch in leaves. It is proposed that expression of AT beta-Amy is regulated by a combination of both positive and negative factors, dependent on the level of sugars, and that HBA1 might function to maintain low-level expression of AT beta-Amy until the level of sugars reaches some high level. Results of crosses of hba1 plants with transgenic plants that harbored an AT beta-Amy:GUS transgene with 1587 bp of the 5'-upstream region suggested that HBA1 affects expressions of AT beta-Amy in trans. The hba1 plants also had growth defects and elevated levels of anthocyanin in their petioles. However, sugar-related changes in levels of several mRNAs other than beta-amylase mRNA were unaffected in hba1 plants, suggesting that only a subset of sugar-regulated genes is under the control HBA1. PMID:9193090

  1. Amylase - urine

    MedlinePlus

    This test is done to diagnose pancreatitis and other diseases that affect the pancreas. ... amylase levels may be a sign of: Acute pancreatitis Alcohol consumption Cancer of the pancreas , ovaries, or ...

  2. The sensitivity and specificity of the RSID-saliva kit for the detection of human salivary amylase in the Forensic Science Laboratory, Dublin, Ireland.

    PubMed

    Casey, David G; Price, Judy

    2010-01-30

    We demonstrate here that the RSID-saliva test can be used as a test for human salivary alpha-amylase on samples routinely examined in forensic casework. We show that the RSID-saliva test detects salivary alpha-amylase at lower concentrations than the Phadebas Quantitative test, that the RSID-saliva test does not cross-react with forensically important human fluids and that the RSID-saliva test can be successfully integrated into the whole swab semen extraction method. PMID:19931992

  3. Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii

    PubMed Central

    Ze, Xiaolei; Ben David, Yonit; Laverde-Gomez, Jenny A.; Dassa, Bareket; Sheridan, Paul O.; Duncan, Sylvia H.; Louis, Petra; Henrissat, Bernard; Juge, Nathalie; Koropatkin, Nicole M.; Bayer, Edward A.

    2015-01-01

    ABSTRACT Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energy from dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches. Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family (GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructose as an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptides were detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted to carry dockerin modules, with one, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediate the formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerins from R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymes Amy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wall-anchoring protein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylases Amy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, as its dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides the first example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.” PMID:26419877

  4. Improvement of cloned [alpha]-amylase gene expression in fed-batch culture of recombinant Saccharomyces cerevisiae by regulating both glucose and ethanol concentrations using a fuzzy controller

    SciTech Connect

    Shiba, Sumihisa; Nishida, Yoshio; Park, Y.S.; Iijima, Shinji; Kobayashi, Takeshi . Dept. of Biotechnology)

    1994-11-05

    The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B-12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the [alpha]-amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. To increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy controller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed-batch culture. The synthesis of [alpha]-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory [alpha]-amylase activities of cells harboring plasmids pNA3 and pNA7 in fed-batch culture were 175 and 392 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled.

  5. Decreased shoot stature and grain alpha-amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat.

    PubMed

    Appleford, Nigel E J; Wilkinson, Mark D; Ma, Qian; Evans, Daniel J; Stone, Marlon C; Pearce, Stephen P; Powers, Stephen J; Thomas, Stephen G; Jones, Huw D; Phillips, Andrew L; Hedden, Peter; Lenton, John R

    2007-01-01

    Ectopic expression of a gibberellin 2-oxidase gene (PcGA2ox1) decreased the content of bioactive gibberellins (GAs) in transgenic wheat, producing a range of dwarf plants with different degrees of severity. In at least one case, a single transformation event gave rise to T(1) plants with different degrees of dwarfism, the phenotypes being stably inherited over at least four generations. The dwarf phenotype, which included dark-green leaves, increased tillering and, in severe cases, a prostrate growth habit, was replicated by the application of a GA biosynthesis inhibitor to the wild type. Ear rachis length, grain set, and grain size were also decreased in the wheat transformants, compared with an azygous (null) line. The extent of post-germination alpha-amylase production in grains reflected the severity of the shoot phenotype of the transformants and both developmental processes were restored to normal by the application of gibberellic acid (GA(3)). Expression of two GA biosynthesis genes (TaGA20ox1 and TaGA3ox2) was up-regulated, and that of two alpha-amylase gene families (alpha-Amy1 and alpha-Amy2) down regulated, in scutella of semi-dwarf lines, compared with controls. The marked decline in transcript abundance of both alpha-amylase gene families in aleurone was associated with a decreased content of bioactive GAs in grains of the semi-dwarf lines. PMID:17916639

  6. Flow injection spectrophotometric analysis of human salivary α-amylase activity using an enzyme degradation of starch-iodine complexes in flow channel and its application to human stress testing.

    PubMed

    Ohtomo, Takao; Igarashi, Shukuro; Takagai, Yoshitaka

    2013-01-01

    Flow injection spectrophotometric analysis (FIA) of human salivary α-amylase was developed using an enzyme degradation reaction of starch-iodine complexes. In this proposed method, the salivary α-amylase, known as a human stress indicator, is directly and rapidly determined without any pretreatment. In this study, the optimum starch-iodine complexes (i.e., optimum molecular weight and amylase-amylopectin compounding ratio) were selected, and their rapid degradation in the flow channel was investigated to determine salivary amylase in the FIA system. The determination range of α-amylase was obtained from 0.25 to 5.0 kilo Novo unit per milliliter (KNU/mL), and these concentrations were equivalent to the real concentration of amylase in human saliva. The quantitative values obtained by this method were found to be highly reproducible with 1.6% (n=25) of the relative standard deviation for 1.0 KNU/mL. The detection limit (3σ) was 60 NU/mL. In addition, the method requires small volume of a sample (20 µL), and 30 samples was sequentially measured within one hour. Real human saliva collected before and after exercise was utilized to demonstrate the feasibility of human stress test and analytical performance of this approach. PMID:24189429

  7. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  8. The impact of single nucleotide polymorphism in monomeric alpha-amylase inhibitor genes from wild emmer wheat, primarily from Israel and Golan

    PubMed Central

    2010-01-01

    Background Various enzyme inhibitors act on key insect gut digestive hydrolases, including alpha-amylases and proteinases. Alpha-amylase inhibitors have been widely investigated for their possible use in strengthening a plant's defense against insects that are highly dependent on starch as an energy source. We attempted to unravel the diversity of monomeric alpha-amylase inhibitor genes of Israeli and Golan Heights' wild emmer wheat with different ecological factors (e.g., geography, water, and temperature). Population methods that analyze the nature and frequency of allele diversity within a species and the codon analysis method (comparing patterns of synonymous and non-synonymous changes in protein coding sequences) were used to detect natural selection. Results Three hundred and forty-eight sequences encoding monomeric alpha-amylase inhibitors (WMAI) were obtained from 14 populations of wild emmer wheat. The frequency of SNPs in WMAI genes was 1 out of 16.3 bases, where 28 SNPs were detected in the coding sequence. The results of purifying and the positive selection hypothesis (p < 0.05) showed that the sequences of WMAI were contributed by both natural selection and co-evolution, which ensured conservation of protein function and inhibition against diverse insect amylases. The majority of amino acid substitutions occurred at the C-terminal (positive selection domain), which ensured the stability of WMAI. SNPs in this gene could be classified into several categories associated with water, temperature, and geographic factors, respectively. Conclusions Great diversity at the WMAI locus, both between and within populations, was detected in the populations of wild emmer wheat. It was revealed that WMAI were naturally selected for across populations by a ratio of dN/dS as expected. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs. A sharp genetic divergence over very short geographic distances compared to

  9. Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601.

    PubMed Central

    Lee, S P; Morikawa, M; Takagi, M; Imanaka, T

    1994-01-01

    A thermophilic and alkaliphilic Bacillus sp. strain, XAL601, was isolated from soil. It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities. The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467). A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found. The deduced amino acid sequence revealed that the four highly conserved regions that are common among amylolytic enzymes were well conserved. These include an active center and common substrate-binding sites of various amylases. In the C-terminal region, a six-amino-acid sequence (Gly-Ser-Gly-Thr-Thr-Pro) is repeated 12 times. The aapT gene was then subcloned in Escherichia coli and overexpressed under the control of the lac promoter. Purification of AapT from this recombinant E. coli was performed, and it was shown that the aapT gene product exhibits both alpha-amylase and pullulanase activities with one active site. The optimum temperature and pH for enzyme activity were found to be 70 degrees C and pH 9, respectively. Furthermore, AapT was found to strongly adsorb to crystalline cellulose (Avicel) and raw corn starch. Final hydrolyzed products from soluble starch range from maltose (G2) to maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. The enzyme also hydrolyzes raw starch under a broad range of conditions (60 to 70 degrees C and pH 8 to 9). Images PMID:7986049

  10. Evolution of the beta-amylase gene in the temperate grasses: Non-purifying selection, recombination, semiparalogy, homeology and phylogenetic signal.

    PubMed

    Minaya, Miguel; Díaz-Pérez, Antonio; Mason-Gamer, Roberta; Pimentel, Manuel; Catalán, Pilar

    2015-10-01

    Low-copy nuclear genes (LCNGs) have complex genetic architectures and evolutionary dynamics. However, unlike multicopy nuclear genes, LCNGs are rarely subject to gene conversion or concerted evolution, and they have higher mutation rates than organellar or nuclear ribosomal DNA markers, so they have great potential for improving the robustness of phylogenetic reconstructions at all taxonomic levels. In this study, our first objective is to evaluate the evolutionary dynamics of the LCNG β-amylase by testing for potential pseudogenization, paralogy, homeology, recombination, and phylogenetic incongruence within a broad representation of the main Pooideae lineages. Our second objective is to determine whether β-amylase shows sufficient phylogenetic signal to reconstruct the evolutionary history of the Pooid grasses. A multigenic (ITS, matK, ndhF, trnTL, and trnLF) tree of the study group provided a framework for assessing the β-amylase phylogeny. Eight accessions showed complete absence of selection, suggesting putative pseudogenic copies or other relaxed selection pressures; resolution of Vulpia alopecuros 2x clones indicated its potential (semi) paralogy; and homeologous copies of allopolyploid species Festuca simensis, F. fenas, and F. arundinacea tracked their Mediterranean origin. Two recombination events were found within early-diverged Pooideae lineages, and five within the PACCMAD clade. The unexpected phylogenetic relationships of 37 grass species (26% of the sampled species) highlight the frequent occurrence of non-treelike evolutionary events, so this LCNG should be used with caution as a phylogenetic marker. However, once the pitfalls are identified and removed, the phylogenetic reconstruction of the grasses based on the β-amylase exon+intron positions is optimal at all taxonomic levels. PMID:26032971

  11. Human Gene Therapy: Genes without Frontiers?

    ERIC Educational Resources Information Center

    Simon, Eric J.

    2002-01-01

    Describes the latest advancements and setbacks in human gene therapy to provide reference material for biology teachers to use in their science classes. Focuses on basic concepts such as recombinant DNA technology, and provides examples of human gene therapy such as severe combined immunodeficiency syndrome, familial hypercholesterolemia, and…

  12. Structure of human salivary alpha-amylase at 1.6 A resolution: implications for its role in the oral cavity.

    PubMed

    Ramasubbu, N; Paloth, V; Luo, Y; Brayer, G D; Levine, M J

    1996-05-01

    Salivary alpha-amylase, a major component of human saliva, plays a role in the initial digestion of starch and may be involved in the colonization of bacteria involved in early dental plaque formation. The three-dimensional atomic structure of salivary amylase has been determined to understand the structure-function relationships of this enzyme. This structure was refined to an R value of 18.4% with 496 amino-acid residues, one calcium ion, one chloride ion and 170 water molecules. Salivary amylase folds into a multidomain structure consisting of three domains, A, B and C. Domain A has a (beta/alpha)(8-) barrel structure, domain B has no definite topology and domain C has a Greek-key barrel structure. The Ca(2+) ion is bound to Asnl00, Arg158, Asp167, His201 and three water molecules. The Cl(-) ion is bound to Arg195, Asn298 and Arg337 and one water molecule. The highly mobile glycine-rich loop 304-310 may act as a gateway for substrate binding and be involved in a 'trap-release' mechanism in the hydrolysis of substrates. Strategic placement of calcium and chloride ions, as well as histidine and tryptophan residues may play a role in differentiating between the glycone and aglycone ends of the polysaccharide substrates. Salivary amylase also possesses a suitable site for binding to enamel surfaces and provides potential sites for the binding of bacterial adhesins. PMID:15299664

  13. Intracellular α-Amylase of Streptococcus mutans

    PubMed Central

    Simpson, Christine L.; Russell, Roy R. B.

    1998-01-01

    Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding α-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular α-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single α-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose. PMID:9721315

  14. Intracellular alpha-amylase of Streptococcus mutans.

    PubMed

    Simpson, C L; Russell, R R

    1998-09-01

    Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose. PMID:9721315

  15. Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes.

    PubMed

    Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

    2014-10-01

    High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. PMID:24975239

  16. Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes

    PubMed Central

    Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

    2014-01-01

    High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. PMID:24975239

  17. The dormancy-breaking stimuli "chilling, hypoxia and cyanamide exposure" up-regulate the expression of α-amylase genes in grapevine buds.

    PubMed

    Rubio, Sebastián; Donoso, Amanda; Pérez, Francisco J

    2014-03-15

    It has been suggested that respiratory stress is involved in the mechanism underlying the dormancy-breaking effect of hydrogen cyanamide (H2CN2) and sodium azide in grapevine buds; indeed, reductions in oxygen levels (hypoxia) and inhibitors of respiration promote bud-break in grapevines. In this study, we showed that, hypoxia increased starch hydrolysis soluble sugar consumption and up-regulated the expression of α-amylase genes (Vvα-AMYs) in grapevine buds, suggesting that these biochemical changes induced by hypoxia, may play a relevant role in the release of buds from endodormancy (ED). Three of the four Vvα-AMY genes that are expressed in grapevine buds were up-regulated by hypoxia and a correlation between changes in sugar content and level of Vvα-AMY gene expression during the hypoxia treatment was found, suggesting that soluble sugars mediate the effect of hypoxia on Vvα-AMY gene expression. Exogenous applications of soluble sugars and sugar analogs confirmed this finding and revealed that osmotic stress induces the expression of Vvα-AMY1 and Vvα-AMY3 and that soluble sugars induces Vvα-AMY2 and Vvα-AMY4 gene expression. Interestingly, the plant hormone gibberellic acid (GA3) induced the expression of Vvα-AMY3 and Vvα-AMY4 genes, while dormancy breaking stimuli, chilling and cyanamide exposure, mainly induced the expression of Vvα-AMY1 and Vvα-AMY2 genes, suggesting that these two α-amylase genes might be involved in the release of grapevine buds from the ED. PMID:24594388

  18. Differential expression of two ß-amylase genes (Bmy1 and Bmy2) in developing and mature barley grain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley (Hordeum vulgare L.) endosperm-specific (Bmy1) and ubiquitous (Bmy2) ß-amylase were studied during the late maturation phase of seed development in four genotypes. Sequencing of Bmy2 from genomic DNA revealed six polymorphisms in the introns and two synonymous SNPs in the coding region. Acc...

  19. Molecular characterization of α-amylase from Staphylococcus aureus.

    PubMed

    Lakshmi, Hanumanthu Prasanna; Prasad, Uppu Venkateswara; Yeswanth, Sthanikam; Swarupa, Vimjam; Prasad, Osuru Hari; Narasu, Mangamoori Lakshmi; Sarma, Potukuchi Venkata Gurunadha Krishna

    2013-01-01

    Staphylococcus aureus is one of the prominent Gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the successful colonization and infection of host tissue. α-amylase is one of the enzymes secreted by S. aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen in any anatomical locales. In the present study we have cloned, sequenced, expressed and characterized α-amylase gene from S. aureus ATCC12600. The recombinant enzyme has a molecular weight of 58kDa and the kinetics showed Vmax 0.0208±0.033 (mg/ml)/mg/min and Km 10.633±0.737mg/ml. The multiple sequence analysis showed α- amylase of S. aureus exhibited large differences with Bacillus subtilis and Streptococcus bovis. As the crystal structure of S. aureus α- amylase was unavailable, we used homology modelling method to build the structure. The built structure was validated by Ramachandran plot which showed 90% of the residues in the allowed region while no residue was found in the disallowed region and the built structure was close to the crystal structure with Z-Score: -6.85. The structural superimposition studies with α- amylases of Bacillus subtilis and Streptococcus bovis showed distinct differences with RMSD values of 18.158Åand 7.091Å respectively which correlated with enzyme kinetics, indicating α-amylase is different among these bacteria. PMID:23559746

  20. Molecular characterization of α-amylase from Staphylococcus aureus

    PubMed Central

    Lakshmi, Hanumanthu Prasanna; Prasad, Uppu Venkateswara; Yeswanth, Sthanikam; Swarupa, Vimjam; Prasad, Osuru Hari; Narasu, Mangamoori Lakshmi; Sarma, Potukuchi Venkata Gurunadha Krishna

    2013-01-01

    Staphylococcus aureus is one of the prominent Gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the successful colonization and infection of host tissue. α-amylase is one of the enzymes secreted by S. aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen in any anatomical locales. In the present study we have cloned, sequenced, expressed and characterized α-amylase gene from S. aureus ATCC12600. The recombinant enzyme has a molecular weight of 58kDa and the kinetics showed Vmax 0.0208±0.033 (mg/ml)/mg/min and Km 10.633±0.737mg/ml. The multiple sequence analysis showed α- amylase of S. aureus exhibited large differences with Bacillus subtilis and Streptococcus bovis. As the crystal structure of S. aureus α- amylase was unavailable, we used homology modelling method to build the structure. The built structure was validated by Ramachandran plot which showed 90% of the residues in the allowed region while no residue was found in the disallowed region and the built structure was close to the crystal structure with Z-Score: -6.85. The structural superimposition studies with α- amylases of Bacillus subtilis and Streptococcus bovis showed distinct differences with RMSD values of 18.158Åand 7.091Å respectively which correlated with enzyme kinetics, indicating α-amylase is different among these bacteria. PMID:23559746

  1. Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

    PubMed

    Tonoike, A; Hori, Y; Inoue-Murayama, M; Konno, A; Fujita, K; Miyado, M; Fukami, M; Nagasawa, M; Mogi, K; Kikusui, T

    2015-10-01

    A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs. PMID:26358734

  2. Genomic structure of the α-amylase gene in the pearl oyster Pinctada fucata and its expression in response to salinity and food concentration.

    PubMed

    Huang, Guiju; Guo, Yihui; Li, Lu; Fan, Sigang; Yu, Ziniu; Yu, Dahui

    2016-08-01

    Amylase is one of the most important digestive enzymes for phytophagous animals. In this study, the cDNA, genomic DNA, and promoter region of the α-amylase gene of the pearl oyster Pinctada fucata were cloned by using reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends, and genome-walking methods. The full-length cDNA sequence was 1704bp long and consisted of a 5'-untranslated region of 17bp, a 3'-untranslated region of 118bp, and a 1569-bp open reading frame encoding a 522-aa polypeptide with a 20-aa signal peptide. Sequence alignment revealed that P. fucata α-amylase (Pfamy) shared the highest identity (91.6%) with Pinctada maxima. The phylogenetic tree showed that it was closely related to P. maxima, based on the amino acid sequences. The genomic DNA was 10850bp and contained nine exons, eight introns, and a promoter region of 3932bp. Several transcriptional factors such as GATA-1, AP-1, and SP1 were predicted in the promoter region. Quantitative RT-PCR assay indicated that the relative expression level of Pfamy was significantly higher in the digestive gland than in other tissues (gonad, gills, muscle, and mantle) (P<0.001). The expression level at salinity 27‰ was significantly higher than that at other salinities (P<0.05). Expression reached a minimum when the algal food concentration was 16×10(4)cells/mL, which was significantly lower than the level observed at 8×10(4)cells/mL and 20×10(4) cells/mL (P<0.05). Our findings provide a genetic basis for further research on Pfamy activity and will facilitate studies on the growth mechanisms and genetic improvement of the pearl oyster P. fucata. PMID:27129943

  3. Evolutionary relationships and sequence variation of alpha-amylase variants encoded by duplicated genes in the Amy locus of Drosophila melanogaster.

    PubMed

    Inomata, N; Shibata, H; Okuyama, E; Yamazaki, T

    1995-09-01

    To infer the genealogical relationships of alpha-amylase electromorphs of Drosophila melanogaster, we determined the nucleotide sequences of a collection of electromorphs sampled throughout the world. On average there were 1.0 amino acid substitutions between identical electromorphs and 3.9 between different electromorphs, respectively. We found that the evolution of AMY1 through AMY6 electromorphs occurred by sequential accumulation of single amino acid substitutions each causing one charge difference. The nucleotide diversities at synonymous sites within Amy1,Amy2,Amy3,Amy4 and Amy6 were 0.0321, 0.0000, 0.0355, 0.0059 and 0.0030, respectively. We also obtained evidence of genetic exchanges, such as intrachromosomal recombination, interchromosomal recombination or gene conversion, between the two duplicated Amy genes as well as among the alleles. PMID:8536971

  4. Genes, Environment, and Human Behavior.

    ERIC Educational Resources Information Center

    Bloom, Mark V.; Cutter, Mary Ann; Davidson, Ronald; Dougherty, Michael J.; Drexler, Edward; Gelernter, Joel; McCullough, Laurence B.; McInerney, Joseph D.; Murray, Jeffrey C.; Vogler, George P.; Zola, John

    This curriculum module explores genes, environment, and human behavior. This book provides materials to teach about the nature and methods of studying human behavior, raise some of the ethical and public policy dilemmas emerging from the Human Genome Project, and provide professional development for teachers. An extensive Teacher Background…

  5. Production of alpha-amylase by yeast

    SciTech Connect

    Thomse, K.K.

    1987-01-01

    The enzyme alpha-amylase confers to an organism the enzymatic activity for the degradation of polyglucosides with alpha-1,4 glycosidic bonds such as starch and glycogen which are among the major storage compounds in plants and animals. Most alpha-amylases are single polypeptides of molecular weights around 50,000 dalton. They are generally found in the digestive tract of animals and in germinating seeds. Among the products released upon enzymatic degradation of polyglucosides maltose, a sugar that can be utilized as carbon source by yeast, is a major constituent. A cDNA segment complementary to mouse salivary amylase messenger RNA has been inserted into the yeast expression vector pMA56 behind the promoter of the gene encoding alcohol dehydrogenase I of yeast. Yeast transformants harboring plasmids with the normal orientation of the promoter and the mouse amylase cDNA gene produce amylase and release the enzyme in free form into the culture medium. Approximately 90% of the amylase activity is found in the medium. Yeast strains carrying MAL allele and transformed with a plasmid which directed the synthesis of mouse alpha-amylase were tested on plates containing starch and in batch fermentations using different high molecular weight sugars and oligosaccharides as carbon source. The results of these experiments will be discussed. (Refs. 21).

  6. Carbohydrate content of acid alpha-glucosidase (gamma-amylase) from human liver.

    PubMed

    Belen'ky, D M; Mikhajlov, V I; Rosenfeld, E L

    1979-05-01

    The presence of carbohydrates in homogeneous preparations of human liver acid alpha-glucosidase has been established and the carbohydrate content of the enzyme determined. The enzyme was purified with the specific purpose of removing all low-molecular-weight carbohydrates. It was specifically adsorbed on Concanavalin A-Sepharose, eluted with methyl-alpha-D-mannopyranoside and gave a positive reaction with the phenol-sulphuric acid reagent. These facts taken together provide evidence that the enzyme studied is a glycoprotein. The analysis of the carbohydrate content of human liver acid alpha-glucosidase showed that there were 8.3 glucosamine, 13.2 mannose and possibly 3--4 glucose residues per molecule of the enzyme with a molecular weight of 98,000. PMID:376187

  7. Method for using a yeast alpha-amylase promoter

    DOEpatents

    Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.

    2003-04-22

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  8. Expression of β-Amylase from Alfalfa Taproots1

    PubMed Central

    Gana, Joyce A.; Kalengamaliro, Newton E.; Cunningham, Suzanne M.; Volenec, Jeffrey J.

    1998-01-01

    Alfalfa (Medicago sativa L.) roots contain large quantities of β-amylase, but little is known about its role in vivo. We studied this by isolating a β-amylase cDNA and by examining signals that affect its expression. The β-amylase cDNA encoded a 55.95-kD polypeptide with a deduced amino acid sequence showing high similarity to other plant β-amylases. Starch concentrations, β-amylase activities, and β-amylase mRNA levels were measured in roots of alfalfa after defoliation, in suspension-cultured cells incubated in sucrose-rich or -deprived media, and in roots of cold-acclimated germ plasms. Starch levels, β-amylase activities, and β-amylase transcripts were reduced significantly in roots of defoliated plants and in sucrose-deprived cell cultures. β-Amylase transcript was high in roots of intact plants but could not be detected 2 to 8 d after defoliation. β-Amylase transcript levels increased in roots between September and October and then declined 10-fold in November and December after shoots were killed by frost. Alfalfa roots contain greater β-amylase transcript levels compared with roots of sweetclover (Melilotus officinalis L.), red clover (Trifolium pratense L.), and birdsfoot trefoil (Lotus corniculatus L.). Southern analysis indicated that β-amylase is present as a multigene family in alfalfa. Our results show no clear association between β-amylase activity or transcript abundance and starch hydrolysis in alfalfa roots. The great abundance of β-amylase and its unexpected patterns of gene expression and protein accumulation support our current belief that this protein serves a storage function in roots of this perennial species. PMID:9847126

  9. Gene losses during human origins.

    PubMed

    Wang, Xiaoxia; Grus, Wendy E; Zhang, Jianzhi

    2006-03-01

    Pseudogenization is a widespread phenomenon in genome evolution, and it has been proposed to serve as an engine of evolutionary change, especially during human origins (the "less-is-more" hypothesis). However, there has been no comprehensive analysis of human-specific pseudogenes. Furthermore, it is unclear whether pseudogenization itself can be selectively favored and thus play an active role in human evolution. Here we conduct a comparative genomic analysis and a literature survey to identify 80 nonprocessed pseudogenes that were inactivated in the human lineage after its separation from the chimpanzee lineage. Many functions are involved among these genes, with chemoreception and immune response being outstandingly overrepresented, suggesting potential species-specific features in these aspects of human physiology. To explore the possibility of adaptive pseudogenization, we focus on CASPASE12, a cysteinyl aspartate proteinase participating in inflammatory and innate immune response to endotoxins. We provide population genetic evidence that the nearly complete fixation of a null allele at CASPASE12 has been driven by positive selection, probably because the null allele confers protection from severe sepsis. We estimate that the selective advantage of the null allele is about 0.9% and the pseudogenization started shortly before the out-of-Africa migration of modern humans. Interestingly, two other genes related to sepsis were also pseudogenized in humans, possibly by selection. These adaptive gene losses might have occurred because of changes in our environment or genetic background that altered the threat from or response to sepsis. The identification and analysis of human-specific pseudogenes open the door for understanding the roles of gene losses in human origins, and the demonstration that gene loss itself can be adaptive supports and extends the "less-is-more" hypothesis. PMID:16464126

  10. Transcriptional gene silencing in humans.

    PubMed

    Weinberg, Marc S; Morris, Kevin V

    2016-08-19

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

  11. Analysis on evolutionary relationship of amylases from archaea, bacteria and eukaryota.

    PubMed

    Yan, Shaomin; Wu, Guang

    2016-02-01

    Amylase is one of the earliest characterized enzymes and has many applications in clinical and industrial settings. In biotechnological industries, the amylase activity is enhanced through modifying amylase structure and through cloning and expressing targeted amylases in different species. It is important to understand how engineered amylases can survive from generation to generation. This study used phylogenetic and statistical approaches to explore general patterns of amylases evolution, including 3118 α-amylases and 280 β-amylases from archaea, eukaryota and bacteria with fully documented taxonomic lineage. First, the phylogenetic tree was created to analyze the evolution of amylases with focus on individual amylases used in biofuel industry. Second, the average pairwise p-distance was computed for each kingdom, phylum, class, order, family and genus, and its diversity implies multi-time and multi-clan evolution. Finally, the variance was further partitioned into inter-clan variance and intra-clan variance for each taxonomic group, and they represent horizontal and vertical gene transfer. Theoretically, the results show a full picture on the evolution of amylases in manners of vertical and horizontal gene transfer, and multi-time and multi-clan evolution as well. Practically, this study provides the information on the surviving chance of desired amylase in a given taxonomic group, which may potentially enhance the successful rate of cloning and expression of amylase gene in different species. PMID:26745984

  12. A new rice zinc-finger protein binds to the O2S box of the alpha-amylase gene promoter.

    PubMed

    Peng, Rihe; Yao, Quanhong; Xiong, Aisheng; Fan, Huiqin; Li, Xian; Peng, Youliang; Cheng, Zong-Ming; Li, Yi

    2004-07-01

    A putative transcription factor, named RAMY, that binds to the 20-bp O2S sequences of the regulatory region of the Amy2 gene promoter has been identified using the yeast one-hybrid system from a rice library. The full length RAMY cDNA clone encodes a 218-amino acid protein and is homologous to the late embryogenesis-abundant protein (LEA5). In vitro mutagenesis and electrophoretic mobility shift assays confirmed that RAMY can bind with O2S specifically through an unusual zinc finger with a CXCX(4)CX(2)H consensus sequence. Low levels of RAMY mRNAs were detected in rice leaves and roots by Northern blot hybridization. The plant hormone gibberellin (GA) induces expression of both RAMY and Amy2 genes, as performed by Northern blot hybridization, but the increase in RAMY mRNA level occurs prior to that of the Amy2 mRNA level in the GA-treated aleurone tissues. These data suggest that RAMY may act as a trans-acting protein and is probably involved in the GA-induced expression of the rice alpha-amylase gene. PMID:15233790

  13. Fermentation of starch by Klebsiella oxytoca P2, containing plasmids with {alpha}-amylase and pullulanase genes

    SciTech Connect

    Santos, V.L. dos; Araujo, E.F.; Barros, E.G. de; Guimaraes, W.V.

    1999-12-20

    Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12--24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower {alpha}-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.

  14. RegG, a CcpA Homolog, Participates in Regulation of Amylase-Binding Protein A Gene (abpA) Expression in Streptococcus gordonii

    PubMed Central

    Rogers, Jeffrey D.; Scannapieco, Frank A.

    2001-01-01

    The amylase-binding protein A (AbpA) of Streptococcus gordonii was found to be undetectable in supernatants of mid-log-phase cultures containing >1% glucose but abundant in supernatants of cultures made with brain heart infusion (BHI), which contains 0.2% glucose. A 10-fold decrease in the level of abpA mRNA in S. gordonii cells cultured in BHI was noted after the addition of glucose to 1%. Analysis of the abpA sequence revealed a potential catabolite responsive element CRE 153 bp downstream of the putative translational start site. A catabolite control protein A gene (ccpA) homolog from S. gordonii, designated regG, was cloned. A regG mutant strain demonstrated moderately less repression of abpA transcription in the presence of 1% glucose. Diauxic growth with glucose and lactose was not affected in the RegG mutant compared to the wild-type parental strain. These results suggest that while RegG plays a role in abpA expression, other mechanisms of catabolite repression are present. PMID:11344161

  15. Antisense SNF1-related (SnRK1) protein kinase gene represses transient activity of an alpha-amylase (alpha-Amy2) gene promoter in cultured wheat embryos.

    PubMed

    Laurie, Sophie; McKibbin, Rowan S; Halford, Nigel G

    2003-02-01

    A DNA fragment corresponding to part of an SNF1 (sucrose non-fermenting-1)-related protein kinase (SnRK1) transcript was amplified by a polymerase chain reaction (PCR) from a wheat (Triticum aestivum) endosperm cDNA library. It was used to construct a chimaeric gene, pUasSnRKN, comprising a ubiquitin promoter, the SnRK1 PCR product in the antisense orientation and the nopaline synthase (Nos) gene terminator. This construct was used in transient gene expression experiments in cultured wheat embryos together with a series of reporter gene constructs. These included the wheat alpha amylase gene alpha-Amy2 promoter with UidA (Gus) coding region (palpha2GT), rice actin promoter with Gus (pActIDGus), ubiquitin promoter with Gus (pAHC25) and actin promoter with green fluorescent protein (GFP) gene (pAct1Is-GFP1). All of the reporter genes were found to be active when bombarded into scutellae isolated from immature grains at 25 d post-anthesis and incubated on MS medium for 24 h prior to bombardment. However, co-bombardment of palpha2GT with equimolar amounts of pUasSnRKN resulted in no detectable Gus activity, indicating that the antisense SnRK1 construct repressed the alpha-Amy2 promoter. Co-bombardment with pUasSnRKN had no effect on the activity of the other promoters used in the study. A triple bombardment with palpha2GT, pAct1Is-GFP-1 and pUasSnRKN resulted in clear green fluorescence, indicating that the bombarded cells were still viable, but no Gus activity. RT-PCR analysis showed clearly that the antisense SnRK1 gene was expressing. Northern and RT-PCR analyses confirmed that SnRK1 and both alpha-amylase genes, alpha-Amy1 and alpha-Amy2, are expressed in cultured wheat embryos harvested from grain 25 d post-anthesis. Expression of alpha-Amy1 and alpha-Amy2 was up-regulated by sugar starvation. PMID:12554717

  16. Syntenic conservation of HSP70 genes in cattle and humans

    SciTech Connect

    Grosz, M.D.; Womack, J.E.; Skow, L.C. )

    1992-12-01

    A phage library of bovine genomic DNA was screened for hybridization with a human HSP70 cDNA probe, and 21 positive plaques were identified and isolated. Restriction mapping and blot hybridization analysis of DNA from the recombinant plaques demonstrated that the cloned DNAs were derived from three different regions of the bovine genome. Ore region contains two tandemly arrayed HSP70 sequences, designated HSP70-1 and HSP70-2, separated by approximately 8 kb of DNA. Single HSP70 sequences, designated HSP70-3 and HSP70-4, were found in two other genomic regions. Locus-specific probes of unique flanking sequences from representative HSP70 clones were hybridized to restriction endonuclease-digested DNA from bovine-hamster and bovine-mouse somatic cell hybrid panels to determine the chromosomal location of the HSP70 sequences. The probe for the tandemly arrayed HSP70-1 and HSP70-2 sequences mapped to bovine chromosome 23, syntenic with glyoxalase 1, 21 steroid hydroxylase, and major histocompatibility class I loci. HSP70-3 sequences mapped to bovine chromosome 10, syntenic with nucleoside phosphorylase and murine osteosarcoma viral oncogene (v-fos), and HSP70-4 mapped to bovine syntenic group U6, syntenic with amylase 1 and phosphoglucomutase 1. On the basis of these data, the authors propose that bovine HSP70-1,2 are homologous to human HSPA1 and HSPA1L on chromosome 6p21.3, bovine HSP70-3 is the homolog of an unnamed human HSP70 gene on chromosome 14q22-q24, and bovine HSP70-4 is homologous to one of the human HSPA-6,-7 genes on chromosome 1. 34 refs., 2 figs., 1 tab.

  17. Activities of Human Gene Nomenclature Committee

    SciTech Connect

    2002-07-16

    The objective of this project, shared between NIH and DOE, has been and remains to enable the medical genetics communities to use common names for genes that are discovered by different gene hunting groups, in different species. This effort provides consistent gene nomenclature and approved gene symbols to the community at large. This contributes to a uniform and consistent understanding of genomes, particularly the human as well as functional genomics based on comparisons between homologous genes in related species (human and mice).

  18. Biased mutagenesis in the N-terminal region by degenerate oligonucleotide gene shuffling enhances secretory expression of barley alpha-amylase 2 in yeast.

    PubMed

    Fukuda, Kenji; Jensen, Malene H; Haser, Richard; Aghajari, Nushin; Svensson, Birte

    2005-11-01

    Recombinant barley alpha-amylase 1 (rAMY1) and 2 (rAMY2), despite 80% sequence identity, are produced in very different amounts of 1.1 and <0.05 mg/l, respectively, by Saccharomyces cerevisiae strain S150-2B. The low yield of AMY2 practically excludes mutational analysis of structure-function relationships and protein engineering. Since different secretion levels of AMY1/AMY2 chimeras were previously ascribed to the N-terminal sequence, AMY1 residues were combinatorially introduced at the 10 non-conserved positions in His14-Gln49 of AMY2 using degenerate oligonucleotide gene shuffling (DOGS) coupled with homologous recombination in S.cerevisiae strain INVSc1. Activity screening of a partial library of 843 clones selected six having a large halo size on starch plates. Three mutants, F21M/Q44H, A42P/A47S and A42P rAMY2, also gave higher activity than wild-type in liquid culture. Only A42P showed wild-type stability and enzymatic properties. The replacement is located to a beta-->alpha loop 2 that interacts with domain B (beta-->alpha loop 3) protruding from the catalytic (beta/alpha)(8)-barrel. Most remarkably Pichia pastoris strain GS115 secreted 60 mg/l A42P compared with 3 mg/l of wild-type rAMY2. The crystal structure of A42P rAMY2 was solved and found to differ marginally from the AMY2 structure, suggesting that the high A42P yield stems from stabilization of the mature and/or intermediate form owing to the introduced proline residue. Moreover, the G to C substitution for the A42P mutation might have a positive impact on protein translation. PMID:16155115

  19. SusG: A Unique Cell-Membrane-Associated [alpha]-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

    SciTech Connect

    Koropatkin, Nicole M.; Smith, Thomas J.

    2010-09-21

    SusG is an {alpha}-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysis demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.

  20. GeneCards Version 3: the human gene integrator.

    PubMed

    Safran, Marilyn; Dalah, Irina; Alexander, Justin; Rosen, Naomi; Iny Stein, Tsippi; Shmoish, Michael; Nativ, Noam; Bahir, Iris; Doniger, Tirza; Krug, Hagit; Sirota-Madi, Alexandra; Olender, Tsviya; Golan, Yaron; Stelzer, Gil; Harel, Arye; Lancet, Doron

    2010-01-01

    GeneCards (www.genecards.org) is a comprehensive, authoritative compendium of annotative information about human genes, widely used for nearly 15 years. Its gene-centric content is automatically mined and integrated from over 80 digital sources, resulting in a web-based deep-linked card for each of >73,000 human gene entries, encompassing the following categories: protein coding, pseudogene, RNA gene, genetic locus, cluster and uncategorized. We now introduce GeneCards Version 3, featuring a speedy and sophisticated search engine and a revamped, technologically enabling infrastructure, catering to the expanding needs of biomedical researchers. A key focus is on gene-set analyses, which leverage GeneCards' unique wealth of combinatorial annotations. These include the GeneALaCart batch query facility, which tabulates user-selected annotations for multiple genes and GeneDecks, which identifies similar genes with shared annotations, and finds set-shared annotations by descriptor enrichment analysis. Such set-centric features address a host of applications, including microarray data analysis, cross-database annotation mapping and gene-disorder associations for drug targeting. We highlight the new Version 3 database architecture, its multi-faceted search engine, and its semi-automated quality assurance system. Data enhancements include an expanded visualization of gene expression patterns in normal and cancer tissues, an integrated alternative splicing pattern display, and augmented multi-source SNPs and pathways sections. GeneCards now provides direct links to gene-related research reagents such as antibodies, recombinant proteins, DNA clones and inhibitory RNAs and features gene-related drugs and compounds lists. We also portray the GeneCards Inferred Functionality Score annotation landscape tool for scoring a gene's functional information status. Finally, we delineate examples of applications and collaborations that have benefited from the GeneCards suite. Database

  1. GeneCards Version 3: the human gene integrator

    PubMed Central

    Safran, Marilyn; Dalah, Irina; Alexander, Justin; Rosen, Naomi; Iny Stein, Tsippi; Shmoish, Michael; Nativ, Noam; Bahir, Iris; Doniger, Tirza; Krug, Hagit; Sirota-Madi, Alexandra; Olender, Tsviya; Golan, Yaron; Stelzer, Gil; Harel, Arye; Lancet, Doron

    2010-01-01

    GeneCards (www.genecards.org) is a comprehensive, authoritative compendium of annotative information about human genes, widely used for nearly 15 years. Its gene-centric content is automatically mined and integrated from over 80 digital sources, resulting in a web-based deep-linked card for each of >73 000 human gene entries, encompassing the following categories: protein coding, pseudogene, RNA gene, genetic locus, cluster and uncategorized. We now introduce GeneCards Version 3, featuring a speedy and sophisticated search engine and a revamped, technologically enabling infrastructure, catering to the expanding needs of biomedical researchers. A key focus is on gene-set analyses, which leverage GeneCards’ unique wealth of combinatorial annotations. These include the GeneALaCart batch query facility, which tabulates user-selected annotations for multiple genes and GeneDecks, which identifies similar genes with shared annotations, and finds set-shared annotations by descriptor enrichment analysis. Such set-centric features address a host of applications, including microarray data analysis, cross-database annotation mapping and gene-disorder associations for drug targeting. We highlight the new Version 3 database architecture, its multi-faceted search engine, and its semi-automated quality assurance system. Data enhancements include an expanded visualization of gene expression patterns in normal and cancer tissues, an integrated alternative splicing pattern display, and augmented multi-source SNPs and pathways sections. GeneCards now provides direct links to gene-related research reagents such as antibodies, recombinant proteins, DNA clones and inhibitory RNAs and features gene-related drugs and compounds lists. We also portray the GeneCards Inferred Functionality Score annotation landscape tool for scoring a gene’s functional information status. Finally, we delineate examples of applications and collaborations that have benefited from the GeneCards suite

  2. Isomaltose formed by alpha-glucosidases triggers amylase induction in Aspergillus nidulans.

    PubMed

    Kato, Naoki; Murakoshi, Yuriko; Kato, Masashi; Kobayashi, Tetsuo; Tsukagoshi, Norihiro

    2002-10-01

    Among various alpha-glucobioses examined, isomaltose was the most effective inducer for amylase synthesis in Aspergillus nidulans. Amylase induction by maltose was completely inhibited by addition of castanospermine or cycloheximide, while induction by isomaltose was not affected by the inhibitors, suggesting that amylase induction by maltose requires inducible alpha-glucosidases. Disruption of the alpha-glucosidase A gene ( agdA), the alpha-glucosidase B gene ( agdB), or both genes did not abolish maltose-dependent induction, although amylase production induced by maltose decreased about 2-fold in the agdA/ agdB double disruptant, compared with that in the agdB disruptant at all concentrations tested. Upon induction by isomaltose, amylase synthesis was enhanced considerably in the agdB and agdA/ agdB disruptants. Even at 3 nM, isomaltose induced amylase production in the double disruptant, supporting the suggestion that isomaltose is a physiological inducer for amylase. Therefore, maltose must be converted to isomaltose by alpha-glucosidases prior to triggering amylase synthesis, but no specific alpha-glucosidase is required for amylase induction by maltose. Probably any alpha-glucosidases having isomaltose-forming activity, including AgdA and AgdB, may participate in amylase induction by maltose. PMID:12420145

  3. Zebrafish orthologs of human muscular dystrophy genes

    PubMed Central

    Steffen, Leta S; Guyon, Jeffrey R; Vogel, Emily D; Beltre, Rosanna; Pusack, Timothy J; Zhou, Yi; Zon, Leonard I; Kunkel, Louis M

    2007-01-01

    Background Human muscular dystrophies are a heterogeneous group of genetic disorders which cause decreased muscle strength and often result in premature death. There is no known cure for muscular dystrophy, nor have all causative genes been identified. Recent work in the small vertebrate zebrafish Danio rerio suggests that mutation or misregulation of zebrafish dystrophy orthologs can also cause muscular degeneration phenotypes in fish. To aid in the identification of new causative genes, this study identifies and maps zebrafish orthologs for all known human muscular dystrophy genes. Results Zebrafish sequence databases were queried for transcripts orthologous to human dystrophy-causing genes, identifying transcripts for 28 out of 29 genes of interest. In addition, the genomic locations of all 29 genes have been found, allowing rapid candidate gene discovery during genetic mapping of zebrafish dystrophy mutants. 19 genes show conservation of syntenic relationships with humans and at least two genes appear to be duplicated in zebrafish. Significant sequence coverage on one or more BAC clone(s) was also identified for 24 of the genes to provide better local sequence information and easy updating of genomic locations as the zebrafish genome assembly continues to evolve. Conclusion This resource supports zebrafish as a dystrophy model, suggesting maintenance of all known dystrophy-associated genes in the zebrafish genome. Coupled with the ability to conduct genetic screens and small molecule screens, zebrafish are thus an attractive model organism for isolating new dystrophy-causing genes/pathways and for use in high-throughput therapeutic discovery. PMID:17374169

  4. Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

    2003-04-01

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  5. Gene expression profiling in developing human hippocampus.

    PubMed

    Zhang, Yan; Mei, Pinchao; Lou, Rong; Zhang, Michael Q; Wu, Guanyun; Qiang, Boqin; Zhang, Zhengguo; Shen, Yan

    2002-10-15

    The gene expression profile of developing human hippocampus is of particular interest and importance to neurobiologists devoted to development of the human brain and related diseases. To gain further molecular insight into the developmental and functional characteristics, we analyzed the expression profile of active genes in developing human hippocampus. Expressed sequence tags (ESTs) were selected by sequencing randomly selected clones from an original 3'-directed cDNA library of 150-day human fetal hippocampus, and a digital expression profile of 946 known genes that could be divided into 16 categories was generated. We also used for comparison 14 other expression profiles of related human neural cells/tissues, including human adult hippocampus. To yield more confidence regarding differential expression, a method was applied to attach normalized expression data to genes with a low false-positive rate (<0.05). Finally, hierarchical cluster analysis was used to exhibit related gene expression patterns. Our results are in accordance with anatomical and physiological observations made during the developmental process of the human hippocampus. Furthermore, some novel findings appeared to be unique to our results. The abundant expression of genes for cell surface components and disease-related genes drew our attention. Twenty-four genes are significantly different from adult, and 13 genes might be developing hippocampus-specific candidate genes, including wnt2b and some Alzheimer's disease-related genes. Our results could provide useful information on the ontogeny, development, and function of cells in the human hippocampus at the molecular level and underscore the utility of large-scale, parallel gene expression analyses in the study of complex biological phenomena. PMID:12271469

  6. Gene Conversion in Human Genetic Disease

    PubMed Central

    Chen, Jian-Min; Férec, Claude; Cooper, David N.

    2010-01-01

    Gene conversion is a specific type of homologous recombination that involves the unidirectional transfer of genetic material from a ‘donor’ sequence to a highly homologous ‘acceptor’. We have recently reviewed the molecular mechanisms underlying gene conversion, explored the key part that this process has played in fashioning extant human genes, and performed a meta-analysis of gene-conversion events known to have caused human genetic disease. Here we shall briefly summarize some of the latest developments in the study of pathogenic gene conversion events, including (i) the emerging idea of minimal efficient sequence homology (MESH) for homologous recombination, (ii) the local DNA sequence features that appear to predispose to gene conversion, (iii) a mechanistic comparison of gene conversion and transient hypermutability, and (iv) recently reported examples of pathogenic gene conversion events. PMID:24710102

  7. The Gene Wiki: community intelligence applied to human gene annotation.

    PubMed

    Huss, Jon W; Lindenbaum, Pierre; Martone, Michael; Roberts, Donabel; Pizarro, Angel; Valafar, Faramarz; Hogenesch, John B; Su, Andrew I

    2010-01-01

    Annotating the function of all human genes is a critical, yet formidable, challenge. Current gene annotation efforts focus on centralized curation resources, but it is increasingly clear that this approach does not scale with the rapid growth of the biomedical literature. The Gene Wiki utilizes an alternative and complementary model based on the principle of community intelligence. Directly integrated within the online encyclopedia, Wikipedia, the goal of this effort is to build a gene-specific review article for every gene in the human genome, where each article is collaboratively written, continuously updated and community reviewed. Previously, we described the creation of Gene Wiki 'stubs' for approximately 9000 human genes. Here, we describe ongoing systematic improvements to these articles to increase their utility. Moreover, we retrospectively examine the community usage and improvement of the Gene Wiki, providing evidence of a critical mass of users and editors. Gene Wiki articles are freely accessible within the Wikipedia web site, and additional links and information are available at http://en.wikipedia.org/wiki/Portal:Gene_Wiki. PMID:19755503

  8. 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.

  9. Aberrant Gene Expression in Humans

    PubMed Central

    Yang, Ence; Ji, Guoli; Brinkmeyer-Langford, Candice L.; Cai, James J.

    2015-01-01

    Gene expression as an intermediate molecular phenotype has been a focus of research interest. In particular, studies of expression quantitative trait loci (eQTL) have offered promise for understanding gene regulation through the discovery of genetic variants that explain variation in gene expression levels. Existing eQTL methods are designed for assessing the effects of common variants, but not rare variants. Here, we address the problem by establishing a novel analytical framework for evaluating the effects of rare or private variants on gene expression. Our method starts from the identification of outlier individuals that show markedly different gene expression from the majority of a population, and then reveals the contributions of private SNPs to the aberrant gene expression in these outliers. Using population-scale mRNA sequencing data, we identify outlier individuals using a multivariate approach. We find that outlier individuals are more readily detected with respect to gene sets that include genes involved in cellular regulation and signal transduction, and less likely to be detected with respect to the gene sets with genes involved in metabolic pathways and other fundamental molecular functions. Analysis of polymorphic data suggests that private SNPs of outlier individuals are enriched in the enhancer and promoter regions of corresponding aberrantly-expressed genes, suggesting a specific regulatory role of private SNPs, while the commonly-occurring regulatory genetic variants (i.e., eQTL SNPs) show little evidence of involvement. Additional data suggest that non-genetic factors may also underlie aberrant gene expression. Taken together, our findings advance a novel viewpoint relevant to situations wherein common eQTLs fail to predict gene expression when heritable, rare inter-individual variation exists. The analytical framework we describe, taking into consideration the reality of differential phenotypic robustness, may be valuable for investigating

  10. Human protein kinase CK2 genes.

    PubMed

    Wirkner, U; Voss, H; Lichter, P; Pyerin, W

    1994-01-01

    We have analyzed the genomic structure of human protein kinase CK2. Of the presumably four genes, the gene encoding the regulatory subunit beta and a processed (pseudo)gene of the catalytic subunit alpha have been characterized completely. In addition, a 18.9 kb-long central part of the gene encoding the catalytic subunit alpha has been characterized. The subunit beta gene spans 4.2 kb and is composed of seven exons. Its promoter region shows several features of a "housekeeping gene" and shares common features with the promoter of the regulatory subunit of cAMP-dependent protein kinase. Conforming to the genomic structure, the beta gene transcripts form a band around 1.1 kb. The central part of the subunit alpha gene contains eight exons comprising bases 102 to 824 of the translated region. Within the introns, 16 Alu repeats were identified, some of which arranged in tandems. The structure of both human CK2 coding genes, alpha and beta, is highly conserved. Several introns are located at corresponding positions in the respective genes of the nematode Caenorhabditis elegans. The processed alpha (pseudo)gene has a complete open reading frame and is 99% homologous to the coding region of the CK2 alpha cDNA. Although the gene has a promoter-like upstream region, no transcript could be identified so far. The genomic clones were used for localization in the human genome. The beta gene was mapped to locus 6p21, the alpha gene to locus 20p13 and the alpha (pseudo)gene to locus 11p15. There is no evidence for additional alpha or beta loci in the human genome. PMID:7735323

  11. Estimation of restraint stress in rats using salivary amylase activity.

    PubMed

    Matsuura, Tetsuya; Takimura, Ryo; Yamaguchi, Masaki; Ichinose, Mitsuyuki

    2012-09-01

    The rat is an ideal model animal for studying physical and psychological stresses. Recent human studies have shown that salivary amylase activity is a useful biomarker of stress in our social life. To estimate the usefulness of amylase activity as a biomarker of stress in rats, we analyzed changes in physiological parameters including amylase activity and anatomical variables, which were induced by a mild restraint of paws (10 min, 3 times/week, 9 weeks). The quantities of food and water intake and excretion amount of the stress rats were smaller than those of the control rats during the experimental period (5-13 weeks). The body weight of the stress rats decreased compared with that of the control rats. Moreover, the enlargement of the adrenal gland was confirmed in the stress rats, indicating that the mild restraint caused a chronic stress response. The amylase activities of the stress rats were significantly greater than those of the control rats at 5 weeks of age. However, the amylase activity of the stress rats decreased compared with that of the control rats after 6 weeks of age. These results indicate that amylase activity is increased by acute stress and reduced by chronic stress, which is caused by repeated restraint stress. In conclusion, amylase activity is a useful biomarker of acute and chronic stresses in rats. PMID:22753135

  12. Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy.

    PubMed

    Papapetrou, Eirini P; Schambach, Axel

    2016-04-01

    Genomic safe harbors (GSHs) are sites in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements: (i) function predictably and (ii) do not cause alterations of the host genome posing a risk to the host cell or organism. GSHs are thus ideal sites for transgene insertion whose use can empower functional genetics studies in basic research and therapeutic applications in human gene therapy. Currently, no fully validated GSHs exist in the human genome. Here, we review our formerly proposed GSH criteria and discuss additional considerations on extending these criteria, on strategies for the identification and validation of GSHs, as well as future prospects on GSH targeting for therapeutic applications. In view of recent advances in genome biology, gene targeting technologies, and regenerative medicine, gene insertion into GSHs can potentially catalyze nearly all applications in human gene therapy. PMID:26867951

  13. Proteinaceous alpha-amylase inhibitors.

    PubMed

    Svensson, Birte; Fukuda, Kenji; Nielsen, Peter K; Bønsager, Birgit C

    2004-02-12

    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous alpha-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological approaches have been outlined for exploitation of the inhibitory function. PMID:14871655

  14. Detergent-compatible bacterial amylases.

    PubMed

    Niyonzima, Francois N; More, Sunil S

    2014-10-01

    Proteases, lipases, amylases, and cellulases are enzymes used in detergent formulation to improve the detergency. The amylases are specifically supplemented to the detergent to digest starchy stains. Most of the solid and liquid detergents that are currently manufactured contain alkaline enzymes. The advantages of using alkaline enzymes in the detergent formulation are that they aid in removing tough stains and the process is environmentally friendly since they reduce the use of toxic detergent ingredients. Amylases active at low temperature are preferred as the energy consumption gets reduced, and the whole process becomes cost-effective. Most microbial alkaline amylases are used as detergent ingredients. Various reviews report on the production, purification, characterization, and application of amylases in different industry sectors, but there is no specific review on bacterial or fungal alkaline amylases or detergent-compatible amylases. In this mini-review, an overview on the production and property studies of the detergent bacterial amylases is given, and the stability and compatibility of the alkaline bacterial amylases in the presence of the detergents and the detergent components are highlighted. PMID:25129040

  15. Characterization of salivary alpha-amylase binding to Streptococcus sanguis

    SciTech Connect

    Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.; Levine, M.J. )

    1989-09-01

    The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.

  16. Characterization of salivary alpha-amylase binding to Streptococcus sanguis.

    PubMed Central

    Scannapieco, F A; Bergey, E J; Reddy, M S; Levine, M J

    1989-01-01

    The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with 125I-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [125I]alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch. Images PMID:2788139

  17. Alpha-amylase from the Hyperthermophilic Archaeon Thermococcus thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, E. C. M. J.; Pusey, M. L.; Ng, M. L.; Garriott, O. K.

    2003-01-01

    Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments such as hot springs. The ability of survival at extreme conditions has rendered enzymes from extremophiles to be of interest in industrial applications. One approach to producing these extremozymes entails the expression of the enzyme-encoding gene in a mesophilic host such as E.coli. This method has been employed in the effort to produce an alpha-amylase from a hyperthermophile (an organism that displays optimal growth above 80 C) isolated from a hydrothermal vent at the Rainbow vent site in the Atlantic Ocean. alpha-amylases catalyze the hydrolysis of starch to produce smaller sugars and constitute a class of industrial enzymes having approximately 25% of the enzyme market. One application for thermostable alpha-amylases is the starch liquefaction process in which starch is converted into fructose and glucose syrups. The a-amylase encoding gene from the hyperthermophile Thermococcus thioreducens was cloned and sequenced, revealing high similarity with other archaeal hyperthermophilic a-amylases. The gene encoding the mature protein was expressed in E.coli. Initial characterization of this enzyme has revealed an optimal amylolytic activity between 85-90 C and around pH 5.3-6.0.

  18. A heterotetrameric alpha-amylase inhibitor from emmer (Triticum dicoccon Schrank) seeds.

    PubMed

    Capocchi, A; Muccilli, V; Cunsolo, V; Saletti, R; Foti, S; Fontanini, D

    2013-04-01

    Plants have developed a constitutive defense system against pest attacks, which involves the expression of a set of inhibitors acting on heterologous amylases of different origins. Investigating the soluble protein complement of the hulled wheat emmer we have isolated and characterized a heterotetrameric α-amylase inhibitor (ETI). Based on mass spectrometry data, it is an assembly of proteins highly similar to the CM2/CM3/CM16 found in durum wheat. Our data indicate that these proteins can also inhibit exogenous α-amylases in binary assemblies. The calculated dissociation constants (K(i)) for the pancreatic porcine amylase- and human salivary amylase-ETI complexes are similar to those found in durum and soft wheat. Homology modeling of the CM subunits indicate structural similarities with other proteins belonging to the cereal family of trypsin/α-amylase inhibitors; a possible homology modeled structure for a tetrameric assembly of the subunits is proposed. PMID:23320956

  19. The retinoblastoma gene in human pituitary tumors

    SciTech Connect

    Cryns, V.L.; Arnold, A.; Alexander, J.M.; Klibanski, A. )

    1993-09-01

    Functional inactivation of the retinoblastoma (RB) tumor suppressor gene is important in the pathogenesis of many human tumors. Recently, the frequent occurrence of pituitary tumors was reported in mice genetically engineered to have one defective RB allele, a genetic background analogous to that of patients with familial retinoblastoma. The molecular pathogenesis of human pituitary tumors is largely unknown, and the potential role of RB gene inactivation in these neoplasms has not been examined. Consequently, the authors studied 20 human pituitary tumors (12 clinically nonfunctioning tumors, 4 somatotroph adenomas, 2 prolactinomas, and 2 corticotrophy adenomas) for tumor-specific allelic loss of the RB gene using a highly informative polymorphic locus within the gene. Control leukocyte DNA samples from 18 of these 20 patients were heterozygous at this locus, permitting genetic evaluation of their paired tumor specimens. In contrast to the pituitary tumors in the mouse model, none of these 18 human tumors exhibited RB allelic loss. These findings indicate that RB gene inactivation probably does not play an important role in the pathogenesis of common types of human pituitary tumors. 24 refs., 1 fig.

  20. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  1. Probing the role of aromatic residues at the secondary saccharide binding sites of human salivary α-amylase in substrate hydrolysis and bacterial binding

    PubMed Central

    Ragunath, Chandran; Manuel, Suba G.A.; Venkataraman, Venkat; Sait, Hameetha B.R.; Kasinathan, Chinnasamy; Ramasubbu, Narayanan

    2008-01-01

    SUMMARY Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: 1) hydrolysis of starch; 2) binding to hydroxyapatite; and 3) binding to bacteria (e.g. viridans streptococci). Although the active site of HSAmy for starch hydrolysis is well characterized, the regions responsible for the bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one or more of the secondary saccharide binding sites harboring the aromatic residues may play an important role in bacterial binding. To test this hypothesis, the aromatic residues at five secondary binding sites were mutated to alanine to generate six mutants representing either single (W203A, Y276A and W284A), double (Y276A/W284A and W316A/W388A) or multiple (HSAmy-ar; W134A/W203A/Y276A/W284A/W316A/W388A) mutations. The crystal structure of HSAmy-ar was determined at a resolution of 1.5 Å as an acarbose complex and compared with the existing wild type acarbose complex. The wild type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch binding, hydroxyapatite and bacterial binding activities. Our results clearly showed that 1) mutation of aromatic residues does not alter the overall conformation of the molecule; 2) the single or double mutants showed either moderate or minimal changes in both starch and bacterial binding activities activity whereas the HSAmy-ar showed significant reduction in these activities; 3) the starch hydrolytic activity was reduced 10-fold in HSAmy-ar; 4) oligosaccharide hydrolytic activity was reduced in all the mutants but the action pattern was similar to that of the wild type enzyme; and 5) the hydroxyaptite binding was unaffected in HSAmy-ar. These results clearly show that the aromatic residues at the secondary saccharide binding sites in HSAmy play a critical role in bacterial binding and starch

  2. Paper-based α-amylase detector for point-of-care diagnostics.

    PubMed

    Dutta, Satarupa; Mandal, Nilanjan; Bandyopadhyay, Dipankar

    2016-04-15

    We report the fabrication of a paper-sensor for quantitative detection of α-amylase activity in human blood serum. Pieces of filter papers were coated with starch-iodine solution leading to an intense blue coloration on the surface. Dispensing α-amylase solution on the starch-iodine coated paper reduced the intensity of the color because of starch-hydrolysis catalyzed by amylase. The variation in the intensity of the color with the concentration of amylase was estimated in three stages: (i) initially, the paper-surface was illuminated with a light emitting diode, (ii) then, the transmitted (reflected) rays emitted through (from) the paper were collected on a photoresistor, and (iii) the variations in the electrical resistance of the photoresistor were correlated with the amylase concentration in analyte. The resistance of photoresistor decreased monotonically with an increase in amylase concentration because the intensity of the reflected (transmitted) rays collected from (through) the paper increased with reduction in the color intensity on the paper surface. Since a specific bio-reaction was employed to detect the activity of amylase, the sensor was found to be equally efficient in detecting unknown quantities of amylase in human blood serum. The reported sensor has shown the potential to graduate into a point-of-care detection tool for α-amylase. PMID:26655186

  3. Advances in gene technology: Human genetic disorders

    SciTech Connect

    Scott, W.A.; Ahmad, F.; Black, S.; Schultz, J.; Whelan, W.J.

    1984-01-01

    This book discusses the papers presented at the conference on the subject of ''advances in Gene technology: Human genetic disorders''. Molecular biology of various carcinomas and inheritance of metabolic diseases is discussed and technology advancement in diagnosis of hereditary diseases is described. Some of the titles discussed are-Immunoglobulin genes translocation and diagnosis; hemophilia; oncogenes; oncogenic transformations; experimental data on mice, hamsters, birds carcinomas and sarcomas.

  4. Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus

    PubMed Central

    Martos-Sitcha, Juan Antonio; Perdomo-Morales, Rolando; Casuso, Antonio; Montero-Alejo, Vivian; García-Galano, Tsai; Martínez-Rodríguez, Gonzalo; Mancera, Juan Miguel

    2016-01-01

    Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase. PMID:27391425

  5. Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus.

    PubMed

    Rodríguez-Viera, Leandro; Perera, Erick; Martos-Sitcha, Juan Antonio; Perdomo-Morales, Rolando; Casuso, Antonio; Montero-Alejo, Vivian; García-Galano, Tsai; Martínez-Rodríguez, Gonzalo; Mancera, Juan Miguel

    2016-01-01

    Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase. PMID:27391425

  6. alpha-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans.

    PubMed

    Agger, T; Spohr, A B; Nielsen, J

    2001-01-01

    The effect of biomass concentration on the formation of Aspergillus oryzae alpha-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of alpha-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2-12 g dry weight kg(-1). When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzae alpha-amylase gene (creA) was deleted, no significant decrease in the specific rate of alpha-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific alpha-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. PMID:11234963

  7. Establishment of a novel, eco-friendly transgenic pig model using porcine pancreatic amylase promoter-driven fungal cellulase transgenes.

    PubMed

    Lin, Y S; Yang, C C; Hsu, C C; Hsu, J T; Wu, S C; Lin, C J; Cheng, W T K

    2015-02-01

    Competition between humans and livestock for cereal and legume grains makes it challenging to provide economical feeds to livestock animals. Recent increases in corn and soybean prices have had a significant impact on the cost of feed for pig producers. The utilization of byproducts and alternative ingredients in pig diets has the potential to reduce feed costs. Moreover, unlike ruminants, pigs have limited ability to utilize diets with high fiber content because they lack endogenous enzymes capable of breaking down nonstarch polysaccharides into simple sugars. Here, we investigated the feasibility of a transgenic strategy in which expression of the fungal cellulase transgene was driven by the porcine pancreatic amylase promoter in pigs. A 2,488 bp 5'-flanking region of the porcine pancreatic amylase gene was cloned by the genomic walking technique, and its structural features were characterized. Using GFP as a reporter, we found that this region contained promoter activity and had the potential to control heterologous gene expression. Transgenic pigs were generated by pronuclear microinjection. Founders and offspring were identified by PCR and Southern blot analyses. Cellulase mRNA and protein showed tissue-specific expression in the pancreas of F1 generation pigs. Cellulolytic enzyme activity was also identified in the pancreas of transgenic pigs. These results demonstrated the establishment of a tissue-specific promoter of the porcine pancreatic amylase gene. Transgenic pigs expressing exogenous cellulase may represent a way to increase the intake of low-cost, fiber-rich feeds. PMID:25063310

  8. Chromosomal localization of the human fibromodulin gene

    SciTech Connect

    Roughley, P.J.; Sztrolovics, R.; Grover, J.

    1994-09-01

    The identification and mapping of genes is a fundamental step in understanding inherited diseases. This study reports the chromosomal localization of the human gene encoding fibromodulin, a collagen-binding proteoglycan which exhibits a wide distribution in connective tissue extracellular matrices. Attempts to localize the gene utilizing a probe covering the published coding region of the human fibromodulin cDNA were unsuccessful. Thus, in order to obtain an alternate probe, the 3{prime}-untranslated region of the cDNA was cloned utilizing the 3{prime}-RACE protocol. Southern blot analysis of human genomic DNA with probes covering either the coding sequence or the 3{prime}-untranslated region revealed simple patterns, indicative of a single-copy gene. Fluorescence in situ hybridization analysis with the 3{prime}-untranslated region probe resulted in hybridization at two chromosomal regions. The majority of signals were observed at 1q32, but some signals were also observed at 9q34.1. The localization of the fibromodulin gene to chromosome 1 was confirmed by the polymerase chain reaction analysis of genomic DNA from a panel of somatic cell hybrid lines. In addition to allowing the gene localization, cloning of the 3{prime}-untranslated region demonstrates that the human fibromodulin cDNA possesses an insert of approximately 160 base pairs which is not present in the published bovine sequence. The human sequence also possesses a single polyadenylation signal, yielding a 3 kb mRNA which was observed in Northern blotting experiments. These results now provide the necessary information to evaluate the potential role of fibromodulin in genetic disorders of connective tissues.

  9. Transcriptional analysis of human survivin gene expression.

    PubMed Central

    Li, F; Altieri, D C

    1999-01-01

    The preservation of tissue and organ homoeostasis depends on the regulated expression of genes controlling apoptosis (programmed cell death). In this study, we have investigated the basal transcriptional requirements of the survivin gene, an IAP (inhibitor of apoptosis) prominently up-regulated in cancer. Analysis of the 5' flanking region of the human survivin gene revealed the presence of a TATA-less promoter containing a canonical CpG island of approximately 250 nt, three cell cycle dependent elements, one cell cycle homology region and numerous Sp1 sites. PCR-based analysis of human genomic DNA, digested with methylation-sensitive and -insensitive restriction enzymes, indicated that the CpG island was unmethylated in both normal and neoplastic tissues. Primer extension and S1 nuclease mapping of the human survivin gene identified two main transcription start sites at position -72 and within -57/-61 from the initiating ATG. Transfection of cervical carcinoma HeLa cells with truncated or nested survivin promoter-luciferase constructs revealed the presence of both enhancer and repressor sequences and identified a minimal promoter region within the proximal -230 nt of the human survivin gene. Unbiased mutagenesis analysis of the human survivin promoter revealed that targeting the Sp1 sequences at position -171 and -151 abolished basal transcriptional activity by approximately 63-82%. Electrophoretic mobility-shift assay with DNA oligonucleotides confirmed formation of a DNA-protein complex between the survivin Sp1 sequences and HeLa cell extracts in a reaction abolished by mutagenesis of the survivin Sp1 sites. These findings identify the basal transcriptional requirements of survivin gene expression. PMID:10567210

  10. Draft Genome Sequences of 18 Oral Streptococcus Strains That Encode Amylase-Binding Proteins

    PubMed Central

    Sabharwal, Amarpreet; Liao, Yu-Chieh; Lin, Hsin-Hung; Haase, Elaine M.

    2015-01-01

    A number of commensal oral streptococcal species produce a heterogeneous group of proteins that mediate binding of salivary α-amylase. This interaction likely influences streptococcal colonization of the oral cavity. Here, we present draft genome sequences of several strains of oral streptococcal species that bind human salivary amylase. PMID:25999552

  11. Draft genome sequences of 18 oral streptococcus strains that encode amylase-binding proteins.

    PubMed

    Sabharwal, Amarpreet; Liao, Yu-Chieh; Lin, Hsin-Hung; Haase, Elaine M; Scannapieco, Frank A

    2015-01-01

    A number of commensal oral streptococcal species produce a heterogeneous group of proteins that mediate binding of salivary α-amylase. This interaction likely influences streptococcal colonization of the oral cavity. Here, we present draft genome sequences of several strains of oral streptococcal species that bind human salivary amylase. PMID:25999552

  12. Gene targeting in primary human trophoblasts

    PubMed Central

    Rosario, Fredrick J; Sadovsky, Yoel; Jansson, Thomas

    2012-01-01

    Studies in primary human trophoblasts provide critical insights into placental function in normal and complicated pregnancies. Mechanistic studies in these cells require experimental tools to modulate gene expression. Lipid-based methods to transfect primary trophoblasts are fairly simple to use and allow for the efficient delivery of nucleic acids, but potential toxic effects limit these methods. Viral vectors are versatile transfection tools of native trophoblastic or foreign cDNAs, providing high transfection efficiency, low toxicity and stable DNA integration into the trophoblast genome. RNA interference (RNAi), using small interfering RNA (siRNA) or microRNA, constitutes a powerful approach to silence trophoblast genes. However, off-target effects, such as regulation of unintended complementary transcripts, inflammatory responses and saturation of the endogenous RNAi machinery, are significant concerns. Strategies to minimize off-target effects include using multiple individual siRNAs, elimination of pro-inflammatory sequences in the siRNA construct and chemical modification of a nucleotide in the guide strand or of the ribose moiety. Tools for efficient gene targeting in primary human trophoblasts are currently available, albeit not yet extensively validated. These methods are critical for exploring the function of human trophoblast genes and may provide a foundation for the future application of gene therapy that targets placental trophoblasts. PMID:22831880

  13. Human pigmentation genes under environmental selection

    PubMed Central

    2012-01-01

    Genome-wide association studies and comparative genomics have established major loci and specific polymorphisms affecting human skin, hair and eye color. Environmental changes have had an impact on selected pigmentation genes as populations have expanded into different regions of the globe. PMID:23110848

  14. Mapping genes to human chromosome 19

    SciTech Connect

    Connolly, Sarah

    1996-05-01

    For this project, 22 Expressed Sequence Tags (ESTs) were fine mapped to regions of human chromosome 19. An EST is a short DNA sequence that occurs once in the genome and corresponds to a single expressed gene. {sup 32}P-radiolabeled probes were made by polymerase chain reaction for each EST and hybridized to filters containing a chromosome 19-specific cosmid library. The location of the ESTs on the chromosome was determined by the location of the ordered cosmid to which the EST hybridized. Of the 22 ESTs that were sublocalized, 6 correspond to known genes, and 16 correspond to anonymous genes. These localized ESTs may serve as potential candidates for disease genes, as well as markers for future physical mapping.

  15. European approach to the Human Gene Project.

    PubMed

    Ferguson-Smith, M A

    1991-01-01

    In the history of gene mapping, which extends through most of the present century, Europe has played an important role. This has continued during the evolution of the 10 International Human Gene Mapping Workshops that have been held in seven different countries since 1973. Nationally coordinated programs have been a recent development, and several European countries, including the United Kingdom and Italy, have followed the lead of the United States in investing substantial sums of money in research on the human genome. In addition, the European Community has launched a multinational program of research on Human Genome Analysis to complement the various national initiatives. The particular approach in Europe has been to support those in the field by establishing resource centers for distributing biomaterials and accessing databases, by assisting in the training of scientists, and by funding programs of research directed at present needs in both physical and genetic mapping. PMID:1991586

  16. Cloning of the human DNA methyltransferase gene

    SciTech Connect

    Ramchanani, S.K.; Rouleau, J.; Szyf, M.

    1994-09-01

    During the process of carcinogenesis it has been observed that DNA methylation is deregulated. At least two levels of regulation of the mouse DNA MeTase have been shown: at the transcriptional level, via its promoter, and at the post transcriptional level in a cell cycle dependent fashion. The sequence of the complete DNA MeTase gene and identification of the promoter has not yet been reported. Using a probe generated by PCR of the human DNA MeTase cDNA, a human genomic library was screened and a clone of approximately 22 kilobases (kb) was isolated. It was found that this clone contains the complete coding sequence of the DNA MeTase enzyme. Sequence analysis along with restriction enzyme digests have allowed us to construct a partial map of the physical structure of the human DNA MeTase gene. This partial structure has already revealed some interesting aspects related to the genetic evolution of the human DNA MeTase. First, the proposed catalytic domain of the human DNA MeTase is extremely homologous to all other cytosine DNA MeTases, even to those that are found in bacteria, and this catalytic domain is conserved within one complete exon in the human gene. This is very different from the structure of the 5{prime} region of the gene, which is fragmented into numerous little introns and exons. Within one of the small introns that have been identified, a trinucleotide repeat of ATG occurs (9 times in a row), and this repeat is upstream of the proposed start site of translation. Trinucleotide repeat expansion has been shown to be a genetic hot spot for mutation, but even more interesting is the nature of the repeat, ATG, which is the translation start codon; this repeat appears to be in frame with the {open_quotes}normal{close_quotes} coding sequence, the implications being that possible alternative methyltransferases may be translated under certain conditions such as cancer.

  17. Neurospora Mutant Exhibiting Hyperproduction of Amylase and Invertase

    PubMed Central

    Gratzner, Howard; Sheehan, D. N.

    1969-01-01

    A mutant strain of Neurospora crassa has been isolated which is derepressed for amylase and β-fructofuranosidase (invertase). Large amounts of the two enzymes were secreted into the culture medium upon depletion of exogenous carbon source. The resulting increases of the two extracellular enzymes were prevented by actinomycin D, cycloheximide, and glycerol. The starving cells of the mutant strain produced amylase and invertase de novo, as evidenced by incorporation of radioactive amino acids into the enzymes. Preliminary genetic studies indicate that these elevated enzyme levels described are due to a single gene mutation. PMID:5773010

  18. Efficient Production of Optically Pure d-Lactic Acid from Raw Corn Starch by Using a Genetically Modified l-Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain▿

    PubMed Central

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure d-lactic acid from raw corn starch, we constructed l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 α-amylase (AmyA). The resulting strain produced only d-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct d-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct d-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct d-lactic acid fermentation from raw starch. PMID:19011066

  19. Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain.

    PubMed

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch. PMID:19011066

  20. Low serum amylase and obesity, diabetes and metabolic syndrome: A novel interpretation.

    PubMed

    Nakajima, Kei

    2016-03-25

    For the last decade, low serum amylase (hypoamylasemia) has been reported in certain common cardiometabolic conditions such as obesity, diabetes (regardless of type), and metabolic syndrome, all of which appear to have a common etiology of insufficient insulin action due to insulin resistance and/or diminished insulin secretion. Some clinical studies have shown that salivary amylase may be preferentially decreased in obese individuals, whereas others have revealed that pancreatic amylase may be preferentially decreased in diabetic subjects with insulin dependence. Despite this accumulated evidence, the clinical relevance of serum, salivary, and pancreatic amylase and the underlying mechanisms have not been fully elucidated. In recent years, copy number variations (CNVs) in the salivary amylase gene (AMY1), which range more broadly than the pancreatic amylase gene (AMY2A and AMY2B), have been shown to be well correlated with salivary and serum amylase levels. In addition, low CNV of AMY1, indicating low salivary amylase, was associated with insulin resistance, obesity, low taste perception/satiety, and postprandial hyperglycemia through impaired insulin secretion at early cephalic phase. In most populations, insulin-dependent diabetes is less prevalent (minor contribution) compared with insulin-independent diabetes, and obesity is highly prevalent compared with low body weight. Therefore, obesity as a condition that elicits cardiometabolic diseases relating to insulin resistance (major contribution) may be a common determinant for low serum amylase in a general population. In this review, the novel interpretation of low serum, salivary, and pancreas amylase is discussed in terms of major contributions of obesity, diabetes, and metabolic syndrome. PMID:27022442

  1. Low serum amylase and obesity, diabetes and metabolic syndrome: A novel interpretation

    PubMed Central

    Nakajima, Kei

    2016-01-01

    For the last decade, low serum amylase (hypoamylasemia) has been reported in certain common cardiometabolic conditions such as obesity, diabetes (regardless of type), and metabolic syndrome, all of which appear to have a common etiology of insufficient insulin action due to insulin resistance and/or diminished insulin secretion. Some clinical studies have shown that salivary amylase may be preferentially decreased in obese individuals, whereas others have revealed that pancreatic amylase may be preferentially decreased in diabetic subjects with insulin dependence. Despite this accumulated evidence, the clinical relevance of serum, salivary, and pancreatic amylase and the underlying mechanisms have not been fully elucidated. In recent years, copy number variations (CNVs) in the salivary amylase gene (AMY1), which range more broadly than the pancreatic amylase gene (AMY2A and AMY2B), have been shown to be well correlated with salivary and serum amylase levels. In addition, low CNV of AMY1, indicating low salivary amylase, was associated with insulin resistance, obesity, low taste perception/satiety, and postprandial hyperglycemia through impaired insulin secretion at early cephalic phase. In most populations, insulin-dependent diabetes is less prevalent (minor contribution) compared with insulin-independent diabetes, and obesity is highly prevalent compared with low body weight. Therefore, obesity as a condition that elicits cardiometabolic diseases relating to insulin resistance (major contribution) may be a common determinant for low serum amylase in a general population. In this review, the novel interpretation of low serum, salivary, and pancreas amylase is discussed in terms of major contributions of obesity, diabetes, and metabolic syndrome. PMID:27022442

  2. Structure of the human progesterone receptor gene.

    PubMed

    Misrahi, M; Venencie, P Y; Saugier-Veber, P; Sar, S; Dessen, P; Milgrom, E

    1993-11-16

    The complete organization of the human progesterone receptor (hPR) gene has been determined. It spans over 90 kbp and contains eight exons. The first exon encodes the N-terminal part of the receptor. The DNA binding domain is encoded by two exons, each exon corresponding to one zinc finger. The steroid binding domain is encoded by five exons. The nucleotide sequence of 1144 bp of the 5' flanking region has been determined. PMID:8241270

  3. Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.).

    PubMed Central

    Schroeder, H. E.; Gollasch, S.; Moore, A.; Tabe, L. M.; Craig, S.; Hardie, D. C.; Chrispeels, M. J.; Spencer, D.; Higgins, TJV.

    1995-01-01

    Bruchid larvae cause major losses of grain legume crops through-out the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing in the field. We transferred the cDNA encoding the [alpha]-amylase inhibitor ([alpha]-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacterium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The [alpha]-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T5 seed generation, and [alpha]-AI accumulated in the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2% [alpha]-AI. In the T5 seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although [alpha]-AI also inhibits human [alpha]-amylase, cooked peas should not have a negative impact on human energy metabolism. PMID:12228429

  4. Zinc oxide nanoparticles as novel alpha-amylase inhibitors

    NASA Astrophysics Data System (ADS)

    Dhobale, Sandip; Thite, Trupti; Laware, S. L.; Rode, C. V.; Koppikar, Soumya J.; Ghanekar, Ruchika-Kaul; Kale, S. N.

    2008-11-01

    Amylase inhibitors, also known as starch blockers, contain substances that prevent dietary starches from being absorbed by the body via inhibiting breakdown of complex sugars to simpler ones. In this sense, these materials are projected as having potential applications in diabetes control. In this context, we report on zinc oxide nanoparticles as possible alpha-amylase inhibitors. Zinc oxide nanoparticles have been synthesized using soft-chemistry approach and 1-thioglycerol was used as a surfactant to yield polycrystalline nanoparticles of size ˜18 nm, stabilized in wurtzite structure. Conjugation study and structural characterization have been done using x-ray diffraction technique, Fourier transform infrared spectroscopy, UV-visible spectroscopy, and transmission electron microscopy. Cytotoxicity studies on human fibrosarcoma (HT-1080) and skin carcinoma (A-431) cell lines as well as mouse primary fibroblast cells demonstrate that up to a dose of 20 μg/ml, ZnO nanoparticles are nontoxic to the cells. We report for the first time the alpha-amylase inhibitory activity of ZnO nanoparticles wherein an optimum dose of 20 μg/ml was sufficient to exhibit 49% glucose inhibition at neutral pH and 35 °C temperature. This inhibitory activity was similar to that obtained with acarbose (a standard alpha-amylase inhibitor), thereby projecting ZnO nanoparticles as novel alpha-amylase inhibitors.

  5. 21 CFR 862.1070 - Amylase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  6. 21 CFR 862.1070 - Amylase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  7. 21 CFR 862.1070 - Amylase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  8. 21 CFR 862.1070 - Amylase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  9. 21 CFR 862.1070 - Amylase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  10. Structure of the human hexabrachion (tenascin) gene.

    PubMed Central

    Gulcher, J R; Nies, D E; Alexakos, M J; Ravikant, N A; Sturgill, M E; Marton, L S; Stefansson, K

    1991-01-01

    The structure of the gene encoding human hexabrachion (tenascin) has been determined from overlapping clones isolated from a human genomic bacteriophage library. The genomic inserts were characterized by restriction mapping, Southern blot analysis, PCR, and DNA sequencing. The coding region of the hexabrachion gene spans approximately 80 kilobases of DNA and consists of 27 exons separated by 26 introns. The exon-intron structure supports a hypothesis based on the cDNA sequence that the hexabrachion gene is an assembly of DNA modules that are also found elsewhere in the genome. Single exons may encode a module, a portion of a module, or a group of modules. The 15 type III units similar to those found in fibronectin are each encoded either by a single exon or by two exons interrupted by an intron. All type III units known to be spliced out of the smaller forms of the protein are encoded by one exon. The fibrinogen-like domain of 210 amino acids is encoded by five exons. The 14.5 epidermal growth factor-like repeats are all encoded by a single exon. Images PMID:1719530

  11. Two human relaxin genes are on chromosome 9.

    PubMed Central

    Crawford, R J; Hudson, P; Shine, J; Niall, H D; Eddy, R L; Shows, T B

    1984-01-01

    We have recently cloned two different human relaxin gene sequences. One of these (H1) was isolated from a human genomic clone bank and the other (H2) from a cDNA library prepared from human pregnant ovarian tissue. Southern gel analysis of the relaxin genes within the genomes of several unrelated individuals showed that all genomes contained both relaxin genes. Hence it is unlikely (p less than 0.001) that the two relaxin gene sequences are alleles. Rather, it is probable that there are two relaxin genes within the human genome. It is likely that relaxin and insulin genes have evolved from a common ancestral gene by gene duplication, since structural similarities between insulin and relaxin are evident at both the peptide and gene level. To investigate the evolutionary relationship between the two human relaxin genes and the insulin gene, we have determined the chromosomal position of the relaxin genes using mouse/human cell hybrids. We found that the human insulin and relaxin genes are on different chromosomes. Both human relaxin genes are located on the short arm region of chromosome 9. Images Fig. 1. Fig. 2. PMID:6548703

  12. Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch

    PubMed Central

    Haase, Elaine M.; Feng, Xianghui; Pan, Jiachuan; Miecznikowski, Jeffrey C.

    2015-01-01

    Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA− mutant KS1ΩabpA under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO2 in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome c-type protein were downregulated in KS1ΩabpA under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance. PMID:26025889

  13. Dietary Methanol Regulates Human Gene Activity

    PubMed Central

    Komarova, Tatiana V.; Sheshukova, Ekaterina V.; Kosorukov, Vyacheslav S.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling. PMID:25033451

  14. Dietary methanol regulates human gene activity.

    PubMed

    Shindyapina, Anastasia V; Petrunia, Igor V; Komarova, Tatiana V; Sheshukova, Ekaterina V; Kosorukov, Vyacheslav S; Kiryanov, Gleb I; Dorokhov, Yuri L

    2014-01-01

    Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling. PMID:25033451

  15. Structure of the human retinoblastoma gene

    SciTech Connect

    Hong, F.D.; Huang, Hueijen S.; To, Hoang; Young, Lihjiuan S.; Oro, A.; Bookstein, R.; Lee, E.Y.H.P.; Lee, Wenhwa )

    1989-07-01

    Complete inactivation of the human retinoblastoma gene (RB) is believed to be an essential step in tumorigenesis of several different cancers. To provide a framework for understanding inactivation mechanisms, the structure of RB was delineated. The RB transcript is encoded in 27 exons dispersed over about 200 kilobases (kb) of genomic DNA. The length of individual exons ranges from 31 to 1,889 base pairs (bp). The largest intron spans >60 kb and the smallest one has only 80 bp. Deletion of exons 13-17 is frequently observed in various types of tumors, including retinoblastoma, breast cancer, and osteosarcoma, and the presence of a potential hot spot for recombination in the region is predicted. A putative leucine-zipper motif is exclusively encoded by exon 20. The detailed RB structure presented should prove useful in defining potential functional domains of its encoded protein. Transcription of RB is initiated at multiple positions and the sequences surrounding the initiation sites have a high G+C content. A typical upstream TATA box is not present. Localization of the RB promoter region was accomplished by utilizing a heterologous expression system containing a bacterial chloramphenicol acetyltransferase gene. Deletion analysis revealed that a region as small as 70 bp is sufficient for RB promoter activity, similar to other previously characterized G+C-rich gene promoters. Several direct repeats and possible stem-and-loop structures are found in the promoter region.

  16. Monoallelic expression of the human FOXP2 speech gene.

    PubMed

    Adegbola, Abidemi A; Cox, Gerald F; Bradshaw, Elizabeth M; Hafler, David A; Gimelbrant, Alexander; Chess, Andrew

    2015-06-01

    The recent descriptions of widespread random monoallelic expression (RMAE) of genes distributed throughout the autosomal genome indicate that there are more genes subject to RMAE on autosomes than the number of genes on the X chromosome where X-inactivation dictates RMAE of X-linked genes. Several of the autosomal genes that undergo RMAE have independently been implicated in human Mendelian disorders. Thus, parsing the relationship between allele-specific expression of these genes and disease is of interest. Mutations in the human forkhead box P2 gene, FOXP2, cause developmental verbal dyspraxia with profound speech and language deficits. Here, we show that the human FOXP2 gene undergoes RMAE. Studying an individual with developmental verbal dyspraxia, we identify a deletion 3 Mb away from the FOXP2 gene, which impacts FOXP2 gene expression in cis. Together these data suggest the intriguing possibility that RMAE impacts the haploinsufficiency phenotypes observed for FOXP2 mutations. PMID:25422445

  17. Structure of the human retinoblastoma gene.

    PubMed Central

    Hong, F D; Huang, H J; To, H; Young, L J; Oro, A; Bookstein, R; Lee, E Y; Lee, W H

    1989-01-01

    Complete inactivation of the human retinoblastoma gene (RB) is believed to be an essential step in tumorigenesis of several different cancers. To provide a framework for understanding inactivation mechanisms, the structure of RB was delineated. The RB transcript is encoded in 27 exons dispersed over about 200 kilobases (kb) of genomic DNA. The length of individual exons ranges from 31 to 1889 base pairs (bp). The largest intron spans greater than 60 kb and the smallest one has only 80 bp. Deletion of exons 13-17 is frequently observed in various types of tumors, including retinoblastoma, breast cancer, and osteosarcoma, and the presence of a potential "hot spot" for recombination in the region is predicted. A putative "leucine-zipper" motif is exclusively encoded by exon 20. The detailed RB structure presented here should prove useful in defining potential functional domains of its encoded protein. Transcription of RB is initiated at multiple positions and the sequences surrounding the initiation sites have a high G + C content. A typical upstream TATA box is not present. Localization of the RB promoter region was accomplished by utilizing a heterologous expression system containing a bacterial chloramphenicol acetyltransferase gene. Deletion analysis revealed that a region as small as 70 bp is sufficient for RB promoter activity, similar to other previously characterized G + C-rich gene promoters. Several direct repeats and possible stem-and-loop structures are found in the promoter region. No enhancer element was detected within the 7.3 kb of upstream sequence studied. Several features of the RB promoter are reminiscent of the characteristics associated with many "housekeeping" genes, consistent with its ubiquitous expression pattern. Images PMID:2748600

  18. Transcriptional regulation of the human biglycan gene.

    PubMed

    Ungefroren, H; Krull, N B

    1996-06-28

    The small leucine-rich proteoglycan biglycan is involved in several physiological and pathophysiological processes through the ability of its core protein to interact with other extracellular matrix molecules and transforming growth factor-beta (TGF-beta). To learn more about the regulation of biglycan core protein expression, we have cloned and sequenced 1218 base pairs from the 5'-flanking region of the human biglycan gene, demonstrated functional promoter activity, and investigated the molecular mechanisms through which various agents modulate its transcriptional activity. Sequencing revealed the presence of several cis-acting elements including multiple AP-2 sites and interleukin-6 response elements, a NF-kappaB site, a TGF-beta negative element, and an E-box. The TATA and CAAT box-lacking promoter possesses many features of a growth-related gene, e.g. a GC-rich immediate 5' region, many Sp1 sites, and the use of multiple transcriptional start sites. Transient transfections of the tumor cell lines MG-63, SK-UT-1, and T47D with various biglycan 5'-flanking region-luciferase reporter gene constructs showed that the proximal 78 base pairs are sufficient for full promoter activity. Several agents among them interleukin-6, and tumor necrosis factor-alpha. were capable of altering biglycan promoter activity. However, in MG-63 cells, TGF-beta1 failed to increase either activity of the biglycan promoter constructs or specific transcription from the endogenous biglycan gene. Since TGF-beta1 also did not alter the stability of cytoplasmic biglycan mRNA as determined from Northern analysis after inhibition of transcription with 5,6-dichloro-1beta-D-ribofuranosylbenzimidazole, an as yet unidentified nuclear post-transcriptional mechanism was considered responsible for the TGF-beta effect in this cell type. These results might help to elucidate the molecular pathways leading to pathological alterations of biglycan expression observed in atherosclerosis, glomerulonephritis

  19. Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

    PubMed

    Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

    2014-03-01

    (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats. PMID:24319124

  20. Genomic structure of the human caldesmon gene.

    PubMed Central

    Hayashi, K; Yano, H; Hashida, T; Takeuchi, R; Takeda, O; Asada, K; Takahashi, E; Kato, I; Sobue, K

    1992-01-01

    The high molecular weight caldesmon (h-CaD) is predominantly expressed in smooth muscles, whereas the low molecular weight caldesmon (l-CaD) is widely distributed in nonmuscle tissues and cells. The changes in CaD isoform expression are closely correlated with the phenotypic modulation of smooth muscle cells. During a search for isoform diversity of human CaDs, l-CaD cDNAs were cloned from HeLa S3 cells. HeLa l-CaD I is composed of 558 amino acids, whereas 26 amino acids (residues 202-227 for HeLa l-CaD I) are deleted in HeLa l-CaD II. The short amino-terminal sequence of HeLa l-CaDs is different from that of fibroblast (WI-38) l-CaD II and human aorta h-CaD. We have also identified WI-38 l-CaD I, which contains a 26-amino acid insertion relative to WI-38 l-CaD II. To reveal the molecular events of the expressional regulation of the CaD isoforms, the genomic structure of the human CaD gene was determined. The human CaD gene is composed of 14 exons and was mapped to a single locus, 7q33-q34. The 26-amino acid insertion is encoded in exon 4 and is specifically spliced in the mRNAs for both h-CaD and l-CaDs I. Exon 3 is the exon that encodes the central repeating domain specific to h-CaD (residues 208-436) together with the common domain in all CaD (residues 73-207 for h-CaD and WI-38 l-CaDs, and residues 68-201 for HeLa l-CaDs). The regulation of h- and l-CaD expression is thought to depend on selection of the two 5' splice sites within exon 3. Thus, the change in expression between l-CaD and h-CaD might be caused by this splicing pathway. Images PMID:1465449

  1. Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians.

    PubMed

    Yamaguchi, Rui; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

    2011-02-01

    A moderately halophilic bacterium, Kocuria varians, was found to produce active α-amylase (K. varians α-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed in Escherichia coli as a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor from Streptomyces nitrosporeus, which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch. PMID:20871989

  2. Comprehensive comparative homeobox gene annotation in human and mouse

    PubMed Central

    Wilming, Laurens G.; Boychenko, Veronika; Harrow, Jennifer L.

    2015-01-01

    Homeobox genes are a group of genes coding for transcription factors with a DNA-binding helix-turn-helix structure called a homeodomain and which play a crucial role in pattern formation during embryogenesis. Many homeobox genes are located in clusters and some of these, most notably the HOX genes, are known to have antisense or opposite strand long non-coding RNA (lncRNA) genes that play a regulatory role. Because automated annotation of both gene clusters and non-coding genes is fraught with difficulty (over-prediction, under-prediction, inaccurate transcript structures), we set out to manually annotate all homeobox genes in the mouse and human genomes. This includes all supported splice variants, pseudogenes and both antisense and flanking lncRNAs. One of the areas where manual annotation has a significant advantage is the annotation of duplicated gene clusters. After comprehensive annotation of all homeobox genes and their antisense genes in human and in mouse, we found some discrepancies with the current gene set in RefSeq regarding exact gene structures and coding versus pseudogene locus biotype. We also identified previously un-annotated pseudogenes in the DUX, Rhox and Obox gene clusters, which helped us re-evaluate and update the gene nomenclature in these regions. We found that human homeobox genes are enriched in antisense lncRNA loci, some of which are known to play a role in gene or gene cluster regulation, compared to their mouse orthologues. Of the annotated set of 241 human protein-coding homeobox genes, 98 have an antisense locus (41%) while of the 277 orthologous mouse genes, only 62 protein coding gene have an antisense locus (22%), based on publicly available transcriptional evidence. PMID:26412852

  3. The economics of human gene patents.

    PubMed

    Scherer, Frederic M

    2002-12-01

    The author examines patents on DNA sequences, including data on gene sequence grants issued by the PTO during a 33-month period from 1998 to 2001. Policy supporting patents on DNA sequences and other elemental information that are far "upstream" in the product development pathway is contrasted with the economic bases and rationale for patents to pharmaceuticals, which require a protracted and expensive process of development and testing but that can be relatively cheaply and competitively imitated once they are approved and disclosed. How to allocate appropriately the economic returns among the upstream and downstream inventors is a challenging problem for economic theory, as well as for contemporary biomedical research, and is perhaps most familiarly embodied in licensing and cross-licensing disputes involving "reach-through" and "reach-back" rights. Such disputes can generate enormous transaction costs. They may become increasingly frequent and vexing with respect to the scope and overlap of patent claims on human gene sequences. On the basis of his analyses, the author argues that genome patent claims should be interpreted narrowly. He is particularly concerned with ensuring that the development of new (therapeutic) products is not blocked or retarded by a multiplicity of prior patent claims, but he is pessimistic that the diversity of participants in biotechnology will provide a "sufficient community of interest to organize comprehensive low-royalty cross-licensing" regimes. Accordingly, he suggests mandatory arbitration as one mechanism for resolving such problems. PMID:12480645

  4. Effects of alpha-amylase on in vitro growth of Legionella pneumophila.

    PubMed Central

    Bortner, C A; Miller, R D; Arnold, R R

    1983-01-01

    Sterile parotid saliva inhibited growth of Legionella pneumophila on solid media, and the salivary component involved in this inhibition has been shown to be amylase. Disk diffusion and well plate assays were used to study possible mechanisms for this effect. The amylolytic activity of saliva copurified with inhibitory activity, and both activities were sensitive to proteinase K digestion and heat treatment. In addition, purified alpha-amylase from several sources (bacteria, fungi, porcine pancreas, and human saliva) exhibited similar activity. Incorporation of charcoal or bovine serum albumin into media blocked inhibition by amylase. Replacement of Bacto-Agar with Noble agar (both from Difco Laboratories) prevented growth inhibition in the absence of starch. However, when corn starch was present with Noble agar, amylase-induced growth inhibition occurred. Purification of starch by washing with methanol eliminated some toxic component. The toxic component from starch could be recovered from the methanol wash and inhibited growth of L. pneumophila in the absence of amylase activity. The results suggest that toxic substances exist in media components which may be unmasked during salivary amylase digestion of starch. This effect may explain, in part, the difficulty in recovery of the organism from clinical specimens containing amylase. PMID:6190756

  5. Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

    PubMed Central

    Ghasemi, Amir; Ghafourian, Sobhan; Vafaei, Sedighe; Mohebi, Reza; Farzi, Maryam; Taherikalani, Morovat; Sadeghifard, Nourkhoda

    2015-01-01

    Objectives In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. Methods Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured. Results Amylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system. Conclusion These results indicate that this expression system was appropriate for the production of thermostable α-amylase. PMID:26835242

  6. Molecular evolution of duplicated amylase gene regions in Drosophila melanogaster: evidence of positive selection in the coding regions and selective constraints in the cis-regulatory regions.

    PubMed Central

    Araki, H; Inomata, N; Yamazaki, T

    2001-01-01

    In this study, we randomly sampled Drosophila melanogaster from Japanese and Kenyan natural populations. We sequenced duplicated (proximal and distal) Amy gene regions to test whether the patterns of polymorphism were consistent with neutral molecular evolution. F(st) between the two geographically distant populations, estimated from Amy gene regions, was 0.084, smaller than reported values for other loci, comparing African and Asian populations. Furthermore, little genetic differentiation was found at a microsatellite locus (DROYANETSB) in these samples (G'st = -0.018). The results of several tests (Tajima's, Fu and Li's, and Wall's tests) were not significantly different from neutrality. However, a significantly higher level of fixed replacement substitutions was detected by a modified McDonald and Kreitman test for both populations. This indicates that positive selection occurred during or immediately after the speciation of D. melanogaster. Sliding-window analysis showed that the proximal region 1, a part of the proximal 5' flanking region, was conserved between D. melanogaster and its sibling species, D. simulans. An HKA test was significant when the proximal region 1 was compared with the 5' flanking region of Alcohol dehydrogenase (Adh), indicating a severe selective constraint on the Amy proximal region 1. These results suggest that natural selection has played an important role in the molecular evolution of Amy gene regions in D. melanogaster. PMID:11156987

  7. Improved human disease candidate gene prioritization using mouse phenotype

    PubMed Central

    Chen, Jing; Xu, Huan; Aronow, Bruce J; Jegga, Anil G

    2007-01-01

    Background The majority of common diseases are multi-factorial and modified by genetically and mechanistically complex polygenic interactions and environmental factors. High-throughput genome-wide studies like linkage analysis and gene expression profiling, tend to be most useful for classification and characterization but do not provide sufficient information to identify or prioritize specific disease causal genes. Results Extending on an earlier hypothesis that the majority of genes that impact or cause disease share membership in any of several functional relationships we, for the first time, show the utility of mouse phenotype data in human disease gene prioritization. We study the effect of different data integration methods, and based on the validation studies, we show that our approach, ToppGene , outperforms two of the existing candidate gene prioritization methods, SUSPECTS and ENDEAVOUR. Conclusion The incorporation of phenotype information for mouse orthologs of human genes greatly improves the human disease candidate gene analysis and prioritization. PMID:17939863

  8. Heterologous expression and secretion of Lactobacillus amylovorus alpha-amylase in Leuconostoc citreum.

    PubMed

    Eom, Hyun-Ju; Moon, Jin-Seok; Seo, Eun-Young; Han, Nam Soo

    2009-11-01

    To develop a gene expression system for Leuconostoc genus, construction of expression vector and expression of a heterologus protein in Leuconostoc was performed. Alpha-amylase gene from Lactobacillus amylovorus was cloned into a Leuconostoc cloning vector, pLeuCM, with its own signal peptide. pLeuCMamy was introduced into Leuconostoc citreum CB2567 and a successful expression of alpha-amy gene was confirmed by enzyme activity assays. About 90% of alpha-amylase activity was detected in the culture broth, revealing most of expressed alpha-amylase was secreted out cells. The signal sequence of alpha-amy gene is a good candidate for the secretion of heterologous protein by using Leuconostoc host-vector system. PMID:19618275

  9. Characterization of a Hydrophobic Amylase Inhibitor from Corn (Zea mays) Seeds with Activity Against Amylase from Fusarium verticillioides.

    PubMed

    Figueira, Edson L Z; Hirooka, Elisa Y; Mendiola-Olaya, Elizabeth; Blanco-Labra, Alejandro

    2003-08-01

    ABSTRACT A hydrophobic 19.7-kDa amylase inhibitor (AI) was purified from corn kernels by 95% ethanol extraction and anionic exchange chromatography. The AI has an isoelectric point of 3.6 and was very stable at different pH values and high temperatures, maintaining 47.6% activity after heating to 94 degrees C for 60 min. Amino acid analysis indicated high valine, leucine, glycine, alanine, and glutamic acid/glutamine content, and especially high valine content (41.2 mol%). This inhibitor is not a glycoprotein. It required 30-min preincubation to maximize complex enzyme-inhibitor formation when the amylase from Fusarium verticillioides was tested. The optimal pH of interaction was 6.5. It showed broad-spectrum activity including the following amylases: human saliva, porcine pancreas, F. verticillioides, as well as those from some insects of agricultural importance (Acanthoscelides obtectus, Zabrotes subfasciatus, Sitophilus zeamais, and Prostephanus truncatus). This novel hydrophobic protein not only inhibited the amylase from F. verticillioides but also decreased the conidia germination. Thus, this protein represents an approach to decrease the production of fumonisin in corn, either by using it as a molecular marker to detect fungal resistance or through genetic engineering. PMID:18943857

  10. Characterization of two coleopteran α-amylases and molecular insights into their differential inhibition by synthetic α-amylase inhibitor, acarbose.

    PubMed

    Channale, Sonal M; Bhide, Amey J; Yadav, Yashpal; Kashyap, Garima; Pawar, Pankaj K; Maheshwari, V L; Ramasamy, Sureshkumar; Giri, Ashok P

    2016-07-01

    Post-harvest insect infestation of stored grains makes them unfit for human consumption and leads to severe economic loss. Here, we report functional and structural characterization of two coleopteran α-amylases viz. Callosobruchus chinensis α-amylase (CcAmy) and Tribolium castaneum α-amylase (TcAmy) along with their interactions with proteinaceous and non-proteinaceous α-amylase inhibitors. Secondary structural alignment of CcAmy and TcAmy with other coleopteran α-amylases revealed conserved motifs, active sites, di-sulfide bonds and two point mutations at spatially conserved substrate or inhibitor-binding sites. Homology modeling and molecular docking showed structural differences between these two enzymes. Both the enzymes had similar optimum pH values but differed in their optimum temperature. Overall, pattern of enzyme stabilities were similar under various temperature and pH conditions. Further, CcAmy and TcAmy differed in their substrate affinity and catalytic efficiency towards starch and amylopectin. HPLC analysis detected common amylolytic products like maltose and malto-triose while glucose and malto-tetrose were unique in CcAmy and TcAmy catalyzed reactions respectively. At very low concentrations, wheat α-amylase inhibitor was found to be superior over the acarbose as far as complete inhibition of amylolytic activities of CcAmy and TcAmy was concerned. Mechanism underlying differential amylolytic reaction inhibition by acarbose was discussed. PMID:27132147

  11. Halophilic Amylase from a Moderately Halophilic Micrococcus

    PubMed Central

    Onishi, Hiroshi

    1972-01-01

    A moderately halophilic Micrococcus sp., isolated from unrefined solar salt, produced a considerable amount of extracellular dextrinogenic amylase when cultivated aerobically in media containing 1 to 3 m NaCl. The Micrococcus amylase had maximal activity at pH 6 to 7 in 1.4 to 2 m NaCl or KCl at 50 C. Calcium ion and a high concentration of NaCl or KCl were essential for activity and stability of the amylase. The salt response of the amylase depended greatly on the pH and temperature of the enzyme assay. PMID:5058445

  12. Gene expression in human fungal pathogen Coccidioides immitis changes as arthroconidia differentiate into spherules and mature

    PubMed Central

    2013-01-01

    Background Coccidioides immitis is a dimorphic fungus that causes disease in mammals, including human beings. It grows as a mycelium containing arthroconidia in the soil and in the host arthroconidia differentiates into a unique structure called a spherule. We used a custom open reading frame oligonucleotide microarray to compare the transcriptome of C. immitis mycelia with early (day 2) and late stage (day 8) spherules grown in vitro. All hybridizations were done in quadruplicate and stringent criteria were used to identify significantly differentially expressed genes. Results 22% of C. immitis genes were differentially expressed in either day 2 or day 8 spherules compared to mycelia, and about 12% of genes were differentially expressed comparing the two spherule time points. Oxireductases, including an extracellular superoxide dismutase, were upregulated in spherules and they may be important for defense against oxidative stress. Many signal transduction molecules, including pleckstrin domain proteins, protein kinases and transcription factors were downregulated in day 2 spherules. Several genes involved in sulfur metabolism were downregulated in day 8 spherules compared to day 2 spherules. Transcription of amylase and α (1,3) glucan synthase was upregulated in spherules; these genes have been found to be important for differentiation to yeast in Histoplasma. There were two homologs of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD); transcription of one was up- and the other downregulated. We tested the effect of a 4-HPPD inhibitor, nitisinone, on mycelial and spherule growth and found that it inhibited mycelial but not spherule growth. Conclusions Transcription of many genes was differentially expressed in the process of arthroconidia to spherule conversion and spherule maturation, as would be expected given the magnitude of the morphologic change. The transcription profile of early stage (day 2) spherules was different than late stage (day 8) endosporulating

  13. The Use of Commercially Available Alpha-Amylase Compounds to Inhibit and Remove Staphylococcus aureus Biofilms

    PubMed Central

    Craigen, Bradford; Dashiff, Aliza; Kadouri, Daniel E

    2011-01-01

    Staphylococcus aureus, a versatile human pathogen, is commonly associated with medical device infections. Its capacity to establish and maintain these infections is thought to be related to its ability to form adherent biofilms. In this study, commercially available α-amylase compounds from various biological sources were evaluated for their ability to reduce and prevent biofilm formation of several S. aureus isolates. Our data demonstrates that α-amylase compounds can rapidly detach biofilms of S. aureus, as well as inhibit biofilm formation. Our data also demonstrates that α-amylase compounds have an ability to reduce and disassociate S. aureus cell-aggregates grown in liquid suspension. These findings suggest that commercially available α-amylase compounds could be used in the future to control S. aureus biofilm-related infections. PMID:21760865

  14. Birth of 'human-specific' genes during primate evolution.

    PubMed

    Nahon, Jean-Louis

    2003-07-01

    Humans and other Anthropoids share very similar chromosome structure and genomic sequence as seen in the 98.5% homology at the DNA level between us and Great Apes. However, anatomical and behavioral traits distinguish Homo sapiens from his closest relatives. I review here several recent studies that address the issue by using different approaches: large-scale sequence comparison (first release) between human and chimpanzee, characterization of recent segmental duplications in the human genome and analysis of exemplary gene families. As a major breakthrough in the field, the heretical concept of 'human-specific' genes has recently received some supporting data. In addition, specific chromosomal regions have been mapped that display all the features of 'gene nurseries' and could have played a major role in gene innovation and speciation during primate evolution. A model is proposed that integrates all known molecular mechanisms that can create new genes in the human lineage. PMID:12868609

  15. In-silico human genomics with GeneCards

    PubMed Central

    2011-01-01

    Since 1998, the bioinformatics, systems biology, genomics and medical communities have enjoyed a synergistic relationship with the GeneCards database of human genes (http://www.genecards.org). This human gene compendium was created to help to introduce order into the increasing chaos of information flow. As a consequence of viewing details and deep links related to specific genes, users have often requested enhanced capabilities, such that, over time, GeneCards has blossomed into a suite of tools (including GeneDecks, GeneALaCart, GeneLoc, GeneNote and GeneAnnot) for a variety of analyses of both single human genes and sets thereof. In this paper, we focus on inhouse and external research activities which have been enabled, enhanced, complemented and, in some cases, motivated by GeneCards. In turn, such interactions have often inspired and propelled improvements in GeneCards. We describe here the evolution and architecture of this project, including examples of synergistic applications in diverse areas such as synthetic lethality in cancer, the annotation of genetic variations in disease, omics integration in a systems biology approach to kidney disease, and bioinformatics tools. PMID:22155609

  16. Injury, inflammation and the emergence of human-specific genes.

    PubMed

    Baird, Andrew; Costantini, Todd; Coimbra, Raul; Eliceiri, Brian P

    2016-05-01

    In light of the central role of inflammation in normal wound repair and regeneration, we hypothesize that the preponderance of human-specific genes expressed in human inflammatory cells is commensurate with the genetic versatility of inflammatory response and the emergence of injuries associated with uniquely hominid behaviors, like a bipedal posture and the use of tools, weapons and fire. The hypothesis underscores the need to study human-specific signaling pathways in experimental models of injury and infers that a selection of human-specific genes, driven in part by the response to injury, may have facilitated the emergence of multifunctional genes expressed in other tissues. PMID:26874655

  17. Human metallothionein genes are clustered on chromosome 16.

    PubMed Central

    Karin, M; Eddy, R L; Henry, W M; Haley, L L; Byers, M G; Shows, T B

    1984-01-01

    The metallothioneins are a family of heavy-metal binding proteins of low molecular weight. They function in the regulation of trace metal metabolism and in the protection against toxic heavy metal ions. In man, the metallothioneins are encoded by at least 10-12 genes separated into two groups, MT-I and MT-II. To understand the genomic organization of these genes and their involvement in hereditary disorders of trace metal metabolism, we have determined their chromosomal location. Using human-mouse cell hybrids and hybridization probes derived from cloned and functional human MT1 and MT2 genes, we show that the functional human genes are clustered on human chromosome 16. Analysis of RNA from somatic cell hybrids indicated that hybrids that contained human chromosome 16 expressed both human MT1 and MT2 mRNA, and this expression is regulated by both heavy metal ions and glucocorticoid hormones. Images PMID:6089206

  18. Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris.

    PubMed

    Gandhi, Sivasangkary; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Chor Leow, Thean; Oslan, Siti Nurbaya

    2015-01-01

    Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL(-1) at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg(-1). The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0-8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t₁/₂) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification. PMID:26090417

  19. Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris

    PubMed Central

    Gandhi, Sivasangkary; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Chor Leow, Thean; Oslan, Siti Nurbaya

    2015-01-01

    Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL−1 at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification. PMID:26090417

  20. Reconstruction of a Functional Human Gene Network, with an Application for Prioritizing Positional Candidate Genes

    PubMed Central

    Franke, Lude; Bakel, Harm van; Fokkens, Like; de Jong, Edwin D.; Egmont-Petersen, Michael; Wijmenga, Cisca

    2006-01-01

    Most common genetic disorders have a complex inheritance and may result from variants in many genes, each contributing only weak effects to the disease. Pinpointing these disease genes within the myriad of susceptibility loci identified in linkage studies is difficult because these loci may contain hundreds of genes. However, in any disorder, most of the disease genes will be involved in only a few different molecular pathways. If we know something about the relationships between the genes, we can assess whether some genes (which may reside in different loci) functionally interact with each other, indicating a joint basis for the disease etiology. There are various repositories of information on pathway relationships. To consolidate this information, we developed a functional human gene network that integrates information on genes and the functional relationships between genes, based on data from the Kyoto Encyclopedia of Genes and Genomes, the Biomolecular Interaction Network Database, Reactome, the Human Protein Reference Database, the Gene Ontology database, predicted protein-protein interactions, human yeast two-hybrid interactions, and microarray coexpressions. We applied this network to interrelate positional candidate genes from different disease loci and then tested 96 heritable disorders for which the Online Mendelian Inheritance in Man database reported at least three disease genes. Artificial susceptibility loci, each containing 100 genes, were constructed around each disease gene, and we used the network to rank these genes on the basis of their functional interactions. By following up the top five genes per artificial locus, we were able to detect at least one known disease gene in 54% of the loci studied, representing a 2.8-fold increase over random selection. This suggests that our method can significantly reduce the cost and effort of pinpointing true disease genes in analyses of disorders for which numerous loci have been reported but for which

  1. Attenuated acute salivary α-amylase responses to gustatory stimulation with citric acid in thin children.

    PubMed

    Chen, Long Hui; Yang, Ze Min; Chen, Wei Wen; Lin, Jing; Zhang, Min; Yang, Xiao Rong; Zhao, Ling Bo

    2015-04-14

    Salivary α-amylase (sAA) is responsible for the 'pre-digestion' of starch in the oral cavity and accounts for up to 50 % of salivary protein in human saliva. An accumulating body of literature suggests that sAA is of nutritional importance; however, it is still not clear how sAA is related to individual's nutritional status. Although copy number variations (CNV) of the salivary amylase gene (AMY1) are associated with variation in sAA levels, a significant amount of sAA variation is not explained by AMY1 CNV. To measure sAA responses to gustatory stimulation with citric acid, we used sAA ratio (the ratio of stimulated sAA levels to those of resting sAA) and investigated acute sAA responses to citric acid in children with normal (Normal-BMI, n 22) and low (Low-BMI, n 21) BMI. The AMY1 gene copy number was determined by quantitative PCR. We, for the first time, demonstrated attenuated acute sAA responses (decreased sAA ratio) to gustatory stimulation in Low-BMI (thinness grade 3) children compared with the Normal-BMI children, which suggest that sAA responses to gustatory stimulation may be of nutritional importance. However, child's nutritional status was not directly related to their resting or stimulated sAA levels, and it was not associated with AMY1 gene copy number. Finally, AMY1 CNV might influence, but did not eventually determine, sAA levels in children. PMID:25784372

  2. Thermally Stable Amylases from Antarctic Psychrophilic Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydrolysis of starch in cold environments by psychrophilic species of bacteria is believed to be accomplished through the production of special cold-adapted amylases. These amylases are reportedly thermally labile with low (<40 deg C) temperature optima and high specific activities at 0 to 25 deg C....

  3. Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

    PubMed

    Agger, Teit; Petersen, Jesper B; O'Connor, Susan M; Murphy, Rachael L; Kelly, Joan M; Nielsen, Jens

    2002-01-18

    The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation. PMID:11689252

  4. Loss of gene function and evolution of human phenotypes

    PubMed Central

    Oh, Hye Ji; Choi, Dongjin; Goh, Chul Jun; Hahn, Yoonsoo

    2015-01-01

    Humans have acquired many distinct evolutionary traits after the human-chimpanzee divergence. These phenotypes have resulted from genetic changes that occurred in the human genome and were retained by natural selection. Comparative primate genome analyses reveal that loss-of-function mutations are common in the human genome. Some of these gene inactivation events were revealed to be associated with the emergence of advantageous phenotypes and were therefore positively selected and fixed in modern humans (the “less-ismore” hypothesis). Representative cases of human gene inactivation and their functional implications are presented in this review. Functional studies of additional inactive genes will provide insight into the molecular mechanisms underlying acquisition of various human-specific traits. [BMB Reports 2015; 48(7): 373-379] PMID:25887751

  5. DNA sequence evolution of the amylase multigene family in Drosophila pseudoobscura.

    PubMed

    Brown, C J; Aquadro, C F; Anderson, W W

    1990-09-01

    The alpha-Amylase locus in Drosophila pseudoobscura is a multigene family of one, two or three copies on the third chromosome. The nucleotide sequences of the three Amylase genes from a single chromosome of D. pseudoobscura are presented. The three Amylase genes differ at about 0.5% of their nucleotides. Each gene has a putative intron of 71 (Amy1) or 81 (Amy2 and Amy3) bp. In contrast, Drosophila melanogaster Amylase genes do not have an intron. The functional Amy1 gene of D. pseudoobscura differs from the Amy-p1 gene of D. melanogaster at an estimated 13.3% of the 1482 nucleotides in the coding region. The estimated rate of synonymous substitutions is 0.398 +/- 0.043, and the estimated rate of nonsynonymous substitutions is 0.068 +/- 0.008. From the sequence data we infer that Amy2 and Amy3 are more closely related to each other than either is to Amy1. From the pattern of nucleotide substitutions we reason that there is selection against synonymous substitutions within the Amy1 sequence; that there is selection against nonsynonymous substitutions within the Amy2 sequence, or that Amy2 has recently undergone a gene conversion with Amy1; and that Amy3 is nonfunctional and subject to random genetic drift. PMID:1699840

  6. Structure and chromosomal localization of the human renal kallikrein gene

    SciTech Connect

    Evans, B.A.; Yun, Z.X.; Close, J.A.; Tregear, G.W.; Kitamura, N.; Nakanish, S.; Callen, D.F.; Baker, E.; Hyland, V.J.; Sutherland, G.R.; Richards, R.I.

    1988-05-03

    Glandular kallikreins are a family of proteases encoded by a variable number of genes in different mammalian species. In all species examined, however, one particular kallikrein is functionally conserved in its capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. This kallikrein is found in the kidney, pancreas, and salivary gland, showing a unique pattern of tissue-specific expression relative to other members of the family. The authors have isolated a genomic clone carrying the human renal kallikrein gene and compared the nucleotide sequence of its promoter region with those of the mouse renal kallikrein gene and another mouse kallikrein gene expressed in a distinct cell type. They find four sequence elements conserved between renal kallikrein genes from the two species. They have also shown that the human gene is localized to 19q13, a position analogous to that of the kallikrein gene family on mouse chromosome 7.

  7. Elevated gene expression levels distinguish human from non-human primate brains

    PubMed Central

    Cáceres, Mario; Lachuer, Joel; Zapala, Matthew A.; Redmond, John C.; Kudo, Lili; Geschwind, Daniel H.; Lockhart, David J.; Preuss, Todd M.; Barlow, Carrolee

    2003-01-01

    Little is known about how the human brain differs from that of our closest relatives. To investigate the genetic basis of human specializations in brain organization and cognition, we compared gene expression profiles for the cerebral cortex of humans, chimpanzees, and rhesus macaques by using several independent techniques. We identified 169 genes that exhibited expression differences between human and chimpanzee cortex, and 91 were ascribed to the human lineage by using macaques as an outgroup. Surprisingly, most differences between the brains of humans and non-human primates involved up-regulation, with ≈90% of the genes being more highly expressed in humans. By contrast, in the comparison of human and chimpanzee heart and liver, the numbers of up- and down-regulated genes were nearly identical. Our results indicate that the human brain displays a distinctive pattern of gene expression relative to non-human primates, with higher expression levels for many genes belonging to a wide variety of functional classes. The increased expression of these genes could provide the basis for extensive modifications of cerebral physiology and function in humans and suggests that the human brain is characterized by elevated levels of neuronal activity. PMID:14557539

  8. Hotspots of Biased Nucleotide Substitutions in Human Genes

    PubMed Central

    Berglund, Jonas; Pollard, Katherine S; Webster, Matthew T

    2009-01-01

    Genes that have experienced accelerated evolutionary rates on the human lineage during recent evolution are candidates for involvement in human-specific adaptations. To determine the forces that cause increased evolutionary rates in certain genes, we analyzed alignments of 10,238 human genes to their orthologues in chimpanzee and macaque. Using a likelihood ratio test, we identified protein-coding sequences with an accelerated rate of base substitutions along the human lineage. Exons evolving at a fast rate in humans have a significant tendency to contain clusters of AT-to-GC (weak-to-strong) biased substitutions. This pattern is also observed in noncoding sequence flanking rapidly evolving exons. Accelerated exons occur in regions with elevated male recombination rates and exhibit an excess of nonsynonymous substitutions relative to the genomic average. We next analyzed genes with significantly elevated ratios of nonsynonymous to synonymous rates of base substitution (dN/dS) along the human lineage, and those with an excess of amino acid replacement substitutions relative to human polymorphism. These genes also show evidence of clusters of weak-to-strong biased substitutions. These findings indicate that a recombination-associated process, such as biased gene conversion (BGC), is driving fixation of GC alleles in the human genome. This process can lead to accelerated evolution in coding sequences and excess amino acid replacement substitutions, thereby generating significant results for tests of positive selection. PMID:19175294

  9. Patenting human genes: when economic interests trump logic and ethics.

    PubMed

    Kluge, Eike-Henner W

    2003-06-01

    To date, over 5,000 applications have been filed with United States Patent Office for patents on human genes. More than 1,500 of these applications have been granted. Other jurisdictions are experiencing a similar rush to mine and protect genomic gold. This paper argues that although many jurisdictions allow the patenting of human genes, this is ethically indefensible and amounts to an unjustified appropriation of a general human heritage. Economic and legal arguments in favour of patenting are considered and rejected. Reference is made to the Wellcome Trust Consortium's initiative and the Merck Gene Index Project, which place patented genetic information into the public domain. PMID:14567475

  10. Differential gene expression in anatomical compartments of the human eye

    PubMed Central

    Diehn, Jennifer J; Diehn, Maximilian; Marmor, Michael F; Brown, Patrick O

    2005-01-01

    Background The human eye is composed of multiple compartments, diverse in form, function, and embryologic origin, that work in concert to provide us with our sense of sight. We set out to systematically characterize the global gene expression patterns that specify the distinctive characteristics of the various eye compartments. Results We used DNA microarrays representing approximately 30,000 human genes to analyze gene expression in the cornea, lens, iris, ciliary body, retina, and optic nerve. The distinctive patterns of expression in each compartment could be interpreted in relation to the physiology and cellular composition of each tissue. Notably, the sets of genes selectively expressed in the retina and in the lens were particularly large and diverse. Genes with roles in immune defense, particularly complement components, were expressed at especially high levels in the anterior segment tissues. We also found consistent differences between the gene expression patterns of the macula and peripheral retina, paralleling the differences in cell layer densities between these regions. Based on the hypothesis that genes responsible for diseases that affect a particular eye compartment are likely to be selectively expressed in that compartment, we compared our gene expression signatures with genetic mapping studies to identify candidate genes for diseases affecting the cornea, lens, and retina. Conclusion Through genome-scale gene expression profiling, we were able to discover distinct gene expression 'signatures' for each eye compartment and identified candidate disease genes that can serve as a reference database for investigating the physiology and pathophysiology of the eye. PMID:16168081

  11. Graphical Features of Functional Genes in Human Protein Interaction Network.

    PubMed

    Wang, Pei; Chen, Yao; Lü, Jinhu; Wang, Qingyun; Yu, Xinghuo

    2016-06-01

    With the completion of the human genome project, it is feasible to investigate large-scale human protein interaction network (HPIN) with complex networks theory. Proteins are encoded by genes. Essential, viable, disease, conserved, housekeeping (HK) and tissue-enriched (TE) genes are functional genes, which are organized and functioned via interaction networks. Based on up-to-date data from various databases or literature, two large-scale HPINs and six subnetworks are constructed. We illustrate that the HPINs and most of the subnetworks are sparse, small-world, scale-free, disassortative and with hierarchical modularity. Among the six subnetworks, essential, disease and HK subnetworks are more densely connected than the others. Statistical analysis on the topological structures of the HPIN reveals that the lethal, the conserved, the HK and the TE genes are with hallmark graphical features. Receiver operating characteristic (ROC) curves indicate that the essential genes can be distinguished from the viable ones with accuracy as high as almost 70%. Closeness, semi-local and eigenvector centralities can distinguish the HK genes from the TE ones with accuracy around 82%. Furthermore, the Venn diagram, cluster dendgrams and classifications of disease genes reveal that some classes of disease genes are with hallmark graphical features, especially for cancer genes, HK disease genes and TE disease genes. The findings facilitate the identification of some functional genes via topological structures. The investigations shed some light on the characteristics of the compete interactome, which have potential implications in networked medicine and biological network control. PMID:26841412

  12. Structure and evolution of the human IKBA gene

    SciTech Connect

    Ito, C.Y.; Bautch, V.L.; Baldwin, A.S. Jr.

    1995-09-20

    I{kappa}B{alpha} belongs to a gene family whose members are characterized by their 6-7 Ankyrin repeats, which allow them to interact with members of the Rel family of transcription factors. We have sequenced a human I{kappa}B{alpha} genomic clone to determine its gene structure. The human I{kappa}B{alpha} gene (IKBA) has six exons and five introns that span approximately 3.5 kb. This genomic organization is similiar to that of other members of the Ankyrin gene family. The human IKBA gene shares similiar intron/exon boundaries with the human BCL3 and NFKB2 genes, which is consistent with their conserved Ankyrin repeats. To examine further the evolutionary relationship between human I{kappa}B{alpha} and other members of its gene family, we performed a phylogenetic analysis. Although the resulting phylogenetic tree does not identify a common ancestor of the I{kappa}B{alpha} gene family, it indicates that this family diverges into two groups based on structure and function. 41 refs., 4 figs., 1 tab.

  13. Nucleotide sequence of the gene for human prothrombin

    SciTech Connect

    Degen, S.J.F.; Davie, E.W.

    1987-09-22

    A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were the compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.

  14. Cellular functions of genetically imprinted genes in human and mouse as annotated in the gene ontology.

    PubMed

    Hamed, Mohamed; Ismael, Siba; Paulsen, Martina; Helms, Volkhard

    2012-01-01

    By analyzing the cellular functions of genetically imprinted genes as annotated in the Gene Ontology for human and mouse, we found that imprinted genes are often involved in developmental, transport and regulatory processes. In the human, paternally expressed genes are enriched in GO terms related to the development of organs and of anatomical structures. In the mouse, maternally expressed genes regulate cation transport as well as G-protein signaling processes. Furthermore, we investigated if imprinted genes are regulated by common transcription factors. We identified 25 TF families that showed an enrichment of binding sites in the set of imprinted genes in human and 40 TF families in mouse. In general, maternally and paternally expressed genes are not regulated by different transcription factors. The genes Nnat, Klf14, Blcap, Gnas and Ube3a contribute most to the enrichment of TF families. In the mouse, genes that are maternally expressed in placenta are enriched for AP1 binding sites. In the human, we found that these genes possessed binding sites for both, AP1 and SP1. PMID:23226257

  15. Genic insights from integrated human proteomics in GeneCards.

    PubMed

    Fishilevich, Simon; Zimmerman, Shahar; Kohn, Asher; Iny Stein, Tsippi; Olender, Tsviya; Kolker, Eugene; Safran, Marilyn; Lancet, Doron

    2016-01-01

    GeneCards is a one-stop shop for searchable human gene annotations (http://www.genecards.org/). Data are automatically mined from ∼120 sources and presented in an integrated web card for every human gene. We report the application of recent advances in proteomics to enhance gene annotation and classification in GeneCards. First, we constructed the Human Integrated Protein Expression Database (HIPED), a unified database of protein abundance in human tissues, based on the publically available mass spectrometry (MS)-based proteomics sources ProteomicsDB, Multi-Omics Profiling Expression Database, Protein Abundance Across Organisms and The MaxQuant DataBase. The integrated database, residing within GeneCards, compares favourably with its individual sources, covering nearly 90% of human protein-coding genes. For gene annotation and comparisons, we first defined a protein expression vector for each gene, based on normalized abundances in 69 normal human tissues. This vector is portrayed in the GeneCards expression section as a bar graph, allowing visual inspection and comparison. These data are juxtaposed with transcriptome bar graphs. Using the protein expression vectors, we further defined a pairwise metric that helps assess expression-based pairwise proximity. This new metric for finding functional partners complements eight others, including sharing of pathways, gene ontology (GO) terms and domains, implemented in the GeneCards Suite. In parallel, we calculated proteome-based differential expression, highlighting a subset of tissues that overexpress a gene and subserving gene classification. This textual annotation allows users of VarElect, the suite's next-generation phenotyper, to more effectively discover causative disease variants. Finally, we define the protein-RNA expression ratio and correlation as yet another attribute of every gene in each tissue, adding further annotative information. The results constitute a significant enhancement of several Gene

  16. Genic insights from integrated human proteomics in GeneCards

    PubMed Central

    Fishilevich, Simon; Zimmerman, Shahar; Kohn, Asher; Iny Stein, Tsippi; Olender, Tsviya; Kolker, Eugene; Safran, Marilyn; Lancet, Doron

    2016-01-01

    GeneCards is a one-stop shop for searchable human gene annotations (http://www.genecards.org/). Data are automatically mined from ∼120 sources and presented in an integrated web card for every human gene. We report the application of recent advances in proteomics to enhance gene annotation and classification in GeneCards. First, we constructed the Human Integrated Protein Expression Database (HIPED), a unified database of protein abundance in human tissues, based on the publically available mass spectrometry (MS)-based proteomics sources ProteomicsDB, Multi-Omics Profiling Expression Database, Protein Abundance Across Organisms and The MaxQuant DataBase. The integrated database, residing within GeneCards, compares favourably with its individual sources, covering nearly 90% of human protein-coding genes. For gene annotation and comparisons, we first defined a protein expression vector for each gene, based on normalized abundances in 69 normal human tissues. This vector is portrayed in the GeneCards expression section as a bar graph, allowing visual inspection and comparison. These data are juxtaposed with transcriptome bar graphs. Using the protein expression vectors, we further defined a pairwise metric that helps assess expression-based pairwise proximity. This new metric for finding functional partners complements eight others, including sharing of pathways, gene ontology (GO) terms and domains, implemented in the GeneCards Suite. In parallel, we calculated proteome-based differential expression, highlighting a subset of tissues that overexpress a gene and subserving gene classification. This textual annotation allows users of VarElect, the suite’s next-generation phenotyper, to more effectively discover causative disease variants. Finally, we define the protein–RNA expression ratio and correlation as yet another attribute of every gene in each tissue, adding further annotative information. The results constitute a significant enhancement of several Gene

  17. Recellularized human dermis for testing gene electrotransfer ex vivo.

    PubMed

    Bulysheva, Anna A; Burcus, Nina; Lundberg, Cathryn; Edelblute, Chelsea M; Francis, Michael P; Heller, Richard

    2016-01-01

    Gene electrotransfer (GET) is a proven and valuable tool for in vivo gene delivery to a variety of tissues such as skin, cardiac muscle, skeletal muscle, and tumors, with controllable gene delivery and expression levels. Optimizing gene expression is a challenging hurdle in preclinical studies, particularly for skin indications, due to differences in electrical conductivity of animal compared to human dermis. Therefore, the goal of this study was to develop an ex vivo model for GET using recellularized human dermis to more closely mimic human skin. Decellularized human dermis (DermACELL(®)) was cultured with human dermal fibroblasts and keratinocytes for 4 weeks. After one week of fibroblast culture, fibroblasts infiltrated and dispersed throughout the dermis. Air-liquid interface culture led to epithelial cell proliferation, stratification and terminal differentiation with distinct basal, spinous, granular and cornified strata. Firefly luciferase expression kinetics were evaluated after GET of recellularized constructs for testing gene delivery parameters to skin in vitro. Elevated luciferase expression persisted up to a week following GET compared to controls without electrotransfer. In summary, recellularized dermis structurally and functionally resembled native human skin in tissue histological organization and homeostasis, proving an effective 3D human skin model for preclinical gene delivery studies. PMID:27121769

  18. Characterization of the human rod transducin alpha-subunit gene.

    PubMed Central

    Fong, S L

    1992-01-01

    The human rod transducin alpha subunit (Tr alpha) gene has been cloned. A cDNA clone, HG14, contained a 1.1 kb insertion when compared with the human Tr alpha cDNA published by Van Dop et al. (1). Based on two overlapping clones isolated from a human genomic library, the human Tr alpha gene is 4.9 kb in length and consists of nine exons interrupted by eight introns. Northern blots of human retina total RNA showed that the gene is transcribed by rod photoreceptors into two species of mRNA, 1.3 kb and 2.4 kb in size. Apparently, this is the result of alternative splicing. Two putative transcription initiation sites were determined by primer extension and S1 nuclease protection assays. The putative promoter regions of the human and mouse Tr alpha genes have an identity of 78.1%. As found in the mouse gene (2), no TATA consensus sequence is present in the human gene. Images PMID:1614872

  19. The smaller human VH gene families display remarkably little polymorphism.

    PubMed Central

    Sanz, I; Kelly, P; Williams, C; Scholl, S; Tucker, P; Capra, J D

    1989-01-01

    We report the nucleotide sequence of 30 distinct human VH gene segments from the VHIV, VHV and VHVI gene families. When these sequences were compared to previously published sequences from these smaller human VH families a surprisingly low level of polymorphism was noted. Two VHIV gene segments from unrelated individuals were identical to two previously published VHIV sequences. Five VHV sequences were identical and seven VHVI gene segments were identical. Where differences were found between the sequences, allele specific oligonucleotide probes were used to verify the germline nature of the change and to test for segregation in several large kindreds. These data provide evidence that at least some human VH gene segments are remarkably stable. Images PMID:2511001

  20. Evolutionary and Topological Properties of Genes and Community Structures in Human Gene Regulatory Networks.

    PubMed

    Szedlak, Anthony; Smith, Nicholas; Liu, Li; Paternostro, Giovanni; Piermarocchi, Carlo

    2016-06-01

    The diverse, specialized genes present in today's lifeforms evolved from a common core of ancient, elementary genes. However, these genes did not evolve individually: gene expression is controlled by a complex network of interactions, and alterations in one gene may drive reciprocal changes in its proteins' binding partners. Like many complex networks, these gene regulatory networks (GRNs) are composed of communities, or clusters of genes with relatively high connectivity. A deep understanding of the relationship between the evolutionary history of single genes and the topological properties of the underlying GRN is integral to evolutionary genetics. Here, we show that the topological properties of an acute myeloid leukemia GRN and a general human GRN are strongly coupled with its genes' evolutionary properties. Slowly evolving ("cold"), old genes tend to interact with each other, as do rapidly evolving ("hot"), young genes. This naturally causes genes to segregate into community structures with relatively homogeneous evolutionary histories. We argue that gene duplication placed old, cold genes and communities at the center of the networks, and young, hot genes and communities at the periphery. We demonstrate this with single-node centrality measures and two new measures of efficiency, the set efficiency and the interset efficiency. We conclude that these methods for studying the relationships between a GRN's community structures and its genes' evolutionary properties provide new perspectives for understanding evolutionary genetics. PMID:27359334

  1. Isolation of a novel amylase and lipase-producing Pseudomonas luteola strain: study of amylase production conditions

    PubMed Central

    2014-01-01

    An amylase and lipase producing bacterium (strain C2) was enriched and isolated from soil regularly contaminated with olive washing wastewater in Sfax, Tunisia. Cell was aerobic, mesophilic, Gram-negative, motile, non-sporulating bacterium, capable of growing optimally at pH 7 and 30°C and tolerated maximally 10% (W/V) NaCl. The predominant fatty acids were found to be C18:1ω7c (32.8%), C16:1ω7c (27.3%) and C16:0 (23.1%). Phylogenetic analysis of the 16S rRNA gene revealed that this strain belonging to the genus Pseudomonas. Strain C2 was found to be closely related to Pseudomonas luteola with more than 99% of similarity. Amylase optimization extraction was carried out using Box Behnken Design (BBD). Its maximal activity was found when the pH and temperature ranged from 5.5 to 6.5 and from 33 to 37°C, respectively. Under these conditions, amylase activity was found to be about 9.48 U/ml. PMID:24405763

  2. Isolation of a novel amylase and lipase-producing Pseudomonas luteola strain: study of amylase production conditions.

    PubMed

    Khannous, Lamia; Jrad, Mouna; Dammak, Mouna; Miladi, Ramzi; Chaaben, Nour; Khemakhem, Bassem; Gharsallah, Néji; Fendri, Imen

    2014-01-01

    An amylase and lipase producing bacterium (strain C2) was enriched and isolated from soil regularly contaminated with olive washing wastewater in Sfax, Tunisia. Cell was aerobic, mesophilic, Gram-negative, motile, non-sporulating bacterium, capable of growing optimally at pH 7 and 30°C and tolerated maximally 10% (W/V) NaCl. The predominant fatty acids were found to be C(18:1)ω7c (32.8%), C(16:1)ω7c (27.3%) and C16:0 (23.1%). Phylogenetic analysis of the 16S rRNA gene revealed that this strain belonging to the genus Pseudomonas. Strain C2 was found to be closely related to Pseudomonas luteola with more than 99% of similarity. Amylase optimization extraction was carried out using Box Behnken Design (BBD). Its maximal activity was found when the pH and temperature ranged from 5.5 to 6.5 and from 33 to 37°C, respectively. Under these conditions, amylase activity was found to be about 9.48 U/ml. PMID:24405763

  3. Human histone gene organization: Nonregular arrangement within a large cluster

    SciTech Connect

    Albig, W.; Meergans, K.; Doenecke, D.

    1997-03-01

    We have previously located the genes of the five human main type H1 genes and the gene encoding the testicular subtype H1t to the region 21.1 to 22.2 on the short arm of chromosome 6. To investigate the organization of the histone genes in this region, we isolated two YACs from a human YAC library by PCR screening with primers specific for histone H1.1. This screen revealed two YAC clones. YAC Y23 (corresponding to ICRFy901D1223) contains an insert of about 480 kb, whereas the smaller YAC 4A (corresponding to ICRFy900C104) spans about 340 kb and is completely covered by YAC Y23. We have subcloned the YAC inserts in cosmids, determined the linear orientation of the cosmids by cosmid walking, and constructed a restriction map of the entire region by mapping the individual cosmids using partial digests and hybridization with labeled oligonucleotides complementary to the cos site of the vector. Hybridization analysis, subcloning, restriction mapping, and sequencing revealed that most of the previously isolated phage and cosmid clones containing histone genes are part of this YAC including the clones containing the four human main type H1 histone genes H1.1 to H1.4, the H1t gene, and core histone genes. Thirty-five histone genes map within 260 kb of the YAC Y23 insert. All newly identified histone genes were sequenced, and the sequences were deposited with the EMBL nucleotide sequence database. The histone H1.5 gene is not part of this region, and we therefore conclude that the H1.5 gene and the associated core histone genes form a separate subcluster within this chromosomal region. 53 refs., 4 figs., 1 tab.

  4. The role of imprinted genes in humans.

    PubMed

    Ishida, Miho; Moore, Gudrun E

    2013-01-01

    Genomic imprinting, a process of epigenetic modification which allows the gene to be expressed in a parent-of-origin specific manner, has an essential role in normal growth and development. Imprinting is found predominantly in placental mammals, and has potentially evolved as a mechanism to balance parental resource allocation to the offspring. Therefore, genetic and epigenetic disruptions which alter the specific dosage of imprinted genes can lead to various developmental abnormalities often associated with fetal growth and neurological behaviour. Over the past 20 years since the first imprinted gene was discovered, many different mechanisms have been implicated in this special regulatory mode of gene expression. This review includes a brief summary of the current understanding of the key molecular events taking place during imprint establishment and maintenance in early embryos, and their relationship to epigenetic disruptions seen in imprinting disorders. Genetic and epigenetic causes of eight recognised imprinting disorders including Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS), and also their association with Assisted reproductive technology (ART) will be discussed. Finally, the role of imprinted genes in fetal growth will be explored by investigating their relationship to a common growth disorder, intrauterine growth restriction (IUGR) and also their potential role in regulating normal growth variation. PMID:22771538

  5. Human Studies of Angiogenic Gene Therapy

    PubMed Central

    Gupta, Rajesh; Tongers, Jörn; Losordo, Douglas W.

    2009-01-01

    Despite significant advances in medical, interventional, and surgical therapy for coronary and peripheral arterial disease, the burden of these illnesses remains high. To address this unmet need, the science of therapeutic angiogenesis has been evolving for almost two decades. Early pre-clinical studies and phase I clinical trials achieved promising results with growth factors administered as recombinant proteins or as single-agent gene therapies, and data accumulated through 10 years of clinical trials indicate that gene therapy has an acceptable safety profile. However, more rigorous phase II and phase III clinical trials have failed to unequivocally demonstrate that angiogenic agents are beneficial under the conditions and in the patients studied to date. Investigators have worked to understand the biology of the vascular system and to incorporate their findings into new treatments for patients with ischemic disease. Recent gene- and cell-therapy trials have demonstrated the bioactivity of several new agents and treatment strategies. Collectively, these observations have renewed interest in the mechanisms of angiogenesis and deepened our understanding of the complexity of vascular regeneration. Gene therapy that incorporates multiple growth factors, approaches that combine cell and gene therapy, and the administration of "master switch" agents that activate numerous downstream pathways are among the credible and plausible steps forward. In this review, we will examine the clinical development of angiogenic therapy, summarize several of the lessons learned during the conduct of these trials, and suggest how this prior experience may guide the conduct of future preclinical investigations and clinical trials. PMID:19815827

  6. Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue.

    PubMed

    Madi, Moinecha; Rols, Marie-Pierre; Gibot, Laure

    2016-01-01

    Gene electrotransfer into the skin is of particular interest for the development of medical applications including DNA vaccination, cancer treatment, wound healing or treatment of local skin disorders. However, such clinical applications are currently limited due to poor understanding of the mechanisms governing DNA electrotransfer within human tissue. Nowadays, most studies are carried out in rodent models but rodent skin varies from human skin in terms of cell composition and architecture. We used a tissue-engineering approach to study gene electrotransfer mechanisms in a human tissue context. Primary human dermal fibroblasts were cultured according to the self-assembly method to produce 3D reconstructed human dermal tissue. In this study, we showed that cells of the reconstructed cutaneous tissue were efficiently electropermeabilized by applying millisecond electric pulses, without affecting their viability. A reporter gene was successfully electrotransferred into this human tissue and gene expression was detected for up to 48h. Interestingly, the transfected cells were solely located on the upper surface of the tissue, where they were in close contact with plasmid DNA solution. Furthermore, we report evidences that electrotransfection success depends on plasmid mobility within tissue- rich in collagens, but not on cell proliferation status. In conclusion, in addition to proposing a reliable alternative to animal experiments, tissue engineering produces valid biological tool for the in vitro study of gene electrotransfer mechanisms in human tissue. PMID:27029947

  7. Novel definition files for human GeneChips based on GeneAnnot

    PubMed Central

    Ferrari, Francesco; Bortoluzzi, Stefania; Coppe, Alessandro; Sirota, Alexandra; Safran, Marilyn; Shmoish, Michael; Ferrari, Sergio; Lancet, Doron; Danieli, Gian Antonio; Bicciato, Silvio

    2007-01-01

    Background Improvements in genome sequence annotation revealed discrepancies in the original probeset/gene assignment in Affymetrix microarray and the existence of differences between annotations and effective alignments of probes and transcription products. In the current generation of Affymetrix human GeneChips, most probesets include probes matching transcripts from more than one gene and probes which do not match any transcribed sequence. Results We developed a novel set of custom Chip Definition Files (CDF) and the corresponding Bioconductor libraries for Affymetrix human GeneChips, based on the information contained in the GeneAnnot database. GeneAnnot-based CDFs are composed of unique custom-probesets, including only probes matching a single gene. Conclusion GeneAnnot-based custom CDFs solve the problem of a reliable reconstruction of expression levels and eliminate the existence of more than one probeset per gene, which often leads to discordant expression signals for the same transcript when gene differential expression is the focus of the analysis. GeneAnnot CDFs are freely distributed and fully compliant with Affymetrix standards and all available software for gene expression analysis. The CDF libraries are available from , along with supplementary information (CDF libraries, installation guidelines and R code, CDF statistics, and analysis results). PMID:18005434

  8. Evaluation of reference genes for gene expression studies in human brown adipose tissue

    PubMed Central

    Taube, Magdalena; Andersson-Assarsson, Johanna C; Lindberg, Kristin; Pereira, Maria J; Gäbel, Markus; Svensson, Maria K; Eriksson, Jan W; Svensson, Per-Arne

    2015-01-01

    Human brown adipose tissue (BAT) has during the last 5 year been subjected to an increasing research interest, due to its putative function as a target for future obesity treatments. The most commonly used method for molecular studies of human BAT is the quantitative polymerase chain reaction (qPCR). This method requires normalization to a reference gene (genes with uniform expression under different experimental conditions, e.g. similar expression levels between human BAT and WAT), but so far no evaluation of reference genes for human BAT has been performed. Two different microarray datasets with samples containing human BAT were used to search for genes with low variability in expression levels. Seven genes (FAM96B, GNB1, GNB2, HUWE1, PSMB2, RING1 and TPT1) identified by microarray analysis, and 8 commonly used reference genes (18S, B2M, GAPDH, LRP10, PPIA, RPLP0, UBC, and YWHAZ) were selected and further analyzed by quantitative PCR in both BAT containing perirenal adipose tissue and subcutaneous adipose tissue. Results were analyzed using 2 different algorithms (Normfinder and geNorm). Most of the commonly used reference genes displayed acceptably low variability (geNorm M-values <0.5) in the samples analyzed, but the novel reference genes identified by microarray displayed an even lower variability (M-values <0.25). Our data suggests that PSMB2, GNB2 and GNB1 are suitable novel reference genes for qPCR analysis of human BAT and we recommend that they are included in future gene expression studies of human BAT. PMID:26451284

  9. Evaluation of reference genes for gene expression studies in human brown adipose tissue.

    PubMed

    Taube, Magdalena; Andersson-Assarsson, Johanna C; Lindberg, Kristin; Pereira, Maria J; Gäbel, Markus; Svensson, Maria K; Eriksson, Jan W; Svensson, Per-Arne

    2015-01-01

    Human brown adipose tissue (BAT) has during the last 5 year been subjected to an increasing research interest, due to its putative function as a target for future obesity treatments. The most commonly used method for molecular studies of human BAT is the quantitative polymerase chain reaction (qPCR). This method requires normalization to a reference gene (genes with uniform expression under different experimental conditions, e.g. similar expression levels between human BAT and WAT), but so far no evaluation of reference genes for human BAT has been performed. Two different microarray datasets with samples containing human BAT were used to search for genes with low variability in expression levels. Seven genes (FAM96B, GNB1, GNB2, HUWE1, PSMB2, RING1 and TPT1) identified by microarray analysis, and 8 commonly used reference genes (18S, B2M, GAPDH, LRP10, PPIA, RPLP0, UBC, and YWHAZ) were selected and further analyzed by quantitative PCR in both BAT containing perirenal adipose tissue and subcutaneous adipose tissue. Results were analyzed using 2 different algorithms (Normfinder and geNorm). Most of the commonly used reference genes displayed acceptably low variability (geNorm M-values <0.5) in the samples analyzed, but the novel reference genes identified by microarray displayed an even lower variability (M-values <0.25). Our data suggests that PSMB2, GNB2 and GNB1 are suitable novel reference genes for qPCR analysis of human BAT and we recommend that they are included in future gene expression studies of human BAT. PMID:26451284

  10. Evolutionary and Topological Properties of Genes and Community Structures in Human Gene Regulatory Networks

    PubMed Central

    Szedlak, Anthony; Smith, Nicholas; Liu, Li; Paternostro, Giovanni; Piermarocchi, Carlo

    2016-01-01

    The diverse, specialized genes present in today’s lifeforms evolved from a common core of ancient, elementary genes. However, these genes did not evolve individually: gene expression is controlled by a complex network of interactions, and alterations in one gene may drive reciprocal changes in its proteins’ binding partners. Like many complex networks, these gene regulatory networks (GRNs) are composed of communities, or clusters of genes with relatively high connectivity. A deep understanding of the relationship between the evolutionary history of single genes and the topological properties of the underlying GRN is integral to evolutionary genetics. Here, we show that the topological properties of an acute myeloid leukemia GRN and a general human GRN are strongly coupled with its genes’ evolutionary properties. Slowly evolving (“cold”), old genes tend to interact with each other, as do rapidly evolving (“hot”), young genes. This naturally causes genes to segregate into community structures with relatively homogeneous evolutionary histories. We argue that gene duplication placed old, cold genes and communities at the center of the networks, and young, hot genes and communities at the periphery. We demonstrate this with single-node centrality measures and two new measures of efficiency, the set efficiency and the interset efficiency. We conclude that these methods for studying the relationships between a GRN’s community structures and its genes’ evolutionary properties provide new perspectives for understanding evolutionary genetics. PMID:27359334

  11. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    PubMed

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  12. Human decorin gene: Intron-exon junctions and chromosomal localization

    SciTech Connect

    Vetter, U.; Young, M.F.; Fisher, L.W. ); Vogel, W.; Just, W. )

    1993-01-01

    All of the protein-encoding exons and the 3[prime]flanking region of the human decorin gene have been cloned an partially sequenced. The locations of the intron-exon junctions within the coding portion of the gene were identical to those found for the homologous human gene, biglycan. The sizes of the introns in the decorin gene, however, were substantially larger than those of the same introns of the biglycan gene. Portions of introns 1, 2, and 3 as well as exon 1 were not found during our extensive screening process. The 5[prime] end of intron 2 was found to have an AG-rich region followed immediately by a CT-rich region. Furthermore, the 5[prime] end of intron 3 was very rich in thymidine, whereas the 3[prime] end of intron 7 was rich in adenosine. Several cDNA clones constructed from cultured human bone cell mRNA were found to contain a different sequence at the 5[prime] end compared to that previously published for mRNA from a human embryonic fibroblast cell line. We were also unable to find the alternate 3[prime] flanking region of the previously published cDNA sequence. We have mapped the human decorin gene by in situ methods to chromosome 12q2l.3. 30 refs., 3 figs., 1 tab.

  13. Transcriptional regulation of human small nuclear RNA genes

    PubMed Central

    Jawdekar, Gauri W.; Henry, R. William

    2009-01-01

    The products of human snRNA genes have been frequently described as performing housekeeping functions and their synthesis refractory to regulation. However, recent studies have emphasized that snRNA and other related non-coding RNA molecules control multiple facets of the central dogma, and their regulated expression is critical to cellular homeostasis during normal growth and in response to stress. Human snRNA genes contain compact and yet powerful promoters that are recognized by increasingly well-characterized transcription factors, thus providing a premier model system to study gene regulation. This review summarizes many recent advances deciphering the mechanism by which the transcription of human snRNA and related genes are regulated. PMID:18442490

  14. Diversity of human copy number variation and multicopy genes.

    PubMed

    Sudmant, Peter H; Kitzman, Jacob O; Antonacci, Francesca; Alkan, Can; Malig, Maika; Tsalenko, Anya; Sampas, Nick; Bruhn, Laurakay; Shendure, Jay; Eichler, Evan E

    2010-10-29

    Copy number variants affect both disease and normal phenotypic variation, but those lying within heavily duplicated, highly identical sequence have been difficult to assay. By analyzing short-read mapping depth for 159 human genomes, we demonstrated accurate estimation of absolute copy number for duplications as small as 1.9 kilobase pairs, ranging from 0 to 48 copies. We identified 4.1 million "singly unique nucleotide" positions informative in distinguishing specific copies and used them to genotype the copy and content of specific paralogs within highly duplicated gene families. These data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association. PMID:21030649

  15. Genes and human behavior: the emerging paradigm.

    PubMed

    Drew, A P

    1997-03-01

    The physical properties of human beings and other organisms as well as their social behavioral traits are manifestations of both genetic inheritance and environment. Recent behavioral research has indicated that certain characteristics or behaviors--such as schizophrenia, divorce, and homosexuality--are highly heritable and are not governed exclusively by social environment. A balanced view of human behavior includes the effects of social learning as well as of genetically determined behavior. A new paradigm promotes enhanced understanding and acceptance of human diversity, be it cultural, racial, or sexual, and has the potential to unite scientists and theologians by creating common grounds of understanding. PMID:15719495

  16. Gene-Environment Interactions in Human Disease: Nuisance or Opportunity?

    PubMed Central

    Ober, Carole; Vercelli, Donata

    2010-01-01

    Many environmental risk factors for common, complex human diseases have been revealed by epidemiologic studies, but how genotypes at specific loci modulate individual responses to environmental risk factors is largely unknown. Gene-environment interactions will be missed in genome-wide association studies and may account for some of the ‘missing heritability’ for these diseases. In this review, we focus on asthma as a model disease for studying gene-environment interactions because of relatively large numbers of candidate gene-environment interactions with asthma risk in the literature. Identifying these interactions using genome-wide approaches poses formidable methodological problems and elucidating molecular mechanisms for these interactions has been challenging. We suggest that studying gene-environment interactions in animal models, while more tractable, is not likely to shed light on the genetic architecture of human diseases. Lastly, we propose avenues for future studies to find gene-environment interactions. PMID:21216485

  17. Novel Primary Immunodeficiency Candidate Genes Predicted by the Human Gene Connectome

    PubMed Central

    Itan, Yuval; Casanova, Jean-Laurent

    2015-01-01

    Germline genetic mutations underlie various primary immunodeficiency (PID) diseases. Patients with rare PID diseases (like most non-PID patients and healthy individuals) carry, on average, 20,000 rare and common coding variants detected by high-throughput sequencing. It is thus a major challenge to select only a few candidate disease-causing variants for experimental testing. One of the tools commonly used in the pipeline for estimating a potential PID-candidate gene is to test whether the specific gene is included in the list of genes that were already experimentally validated as PID-causing in previous studies. However, this approach is limited because it cannot detect the PID-causing mutation(s) in the many PID patients carrying causal mutations of as yet unidentified PID-causing genes. In this study, we expanded in silico the list of potential PID-causing candidate genes from 229 to 3,110. We first identified the top 1% of human genes predicted by the human genes connectome to be biologically close to the 229 known PID genes. We then further narrowed down the list of genes by retaining only the most biologically relevant genes, with functionally enriched gene ontology biological categories similar to those for the known PID genes. We validated this prediction by showing that 17 of the 21 novel PID genes published since the last IUIS classification fall into this group of 3,110 genes (p < 10−7). The resulting new extended list of 3,110 predicted PID genes should be useful for the discovery of novel PID genes in patients. PMID:25883595

  18. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  19. Human cancers overexpress genes that are specific to a variety of normal human tissues

    PubMed Central

    Lotem, Joseph; Netanely, Dvir; Domany, Eytan; Sachs, Leo

    2005-01-01

    We have analyzed gene expression data from three different kinds of samples: normal human tissues, human cancer cell lines, and leukemic cells from lymphoid and myeloid leukemia pediatric patients. We have searched for genes that are overexpressed in human cancer and also show specific patterns of tissue-dependent expression in normal tissues. Using the expression data of the normal tissues, we identified 4,346 genes with a high variability of expression and clustered these genes according to their relative expression level. Of 91 stable clusters obtained, 24 clusters included genes preferentially expressed either only in hematopoietic tissues or in hematopoietic and one to two other tissues; 28 clusters included genes preferentially expressed in various nonhematopoietic tissues such as neuronal, testis, liver, kidney, muscle, lung, pancreas, and placenta. Analysis of the expression levels of these two groups of genes in the human cancer cell lines and leukemias identified genes that were highly expressed in cancer cells but not in their normal counterparts and, thus, were overexpressed in the cancers. The different cancer cell lines and leukemias varied in the number and identity of these overexpressed genes. The results indicate that many genes that are overexpressed in human cancer cells are specific to a variety of normal tissues, including normal tissues other than those from which the cancer originated. It is suggested that this general property of cancer cells plays a major role in determining the behavior of the cancers, including their metastatic potential. PMID:16339305

  20. Complementation of Yeast Genes with Human Genes as an Experimental Platform for Functional Testing of Human Genetic Variants

    PubMed Central

    Hamza, Akil; Tammpere, Erik; Kofoed, Megan; Keong, Christelle; Chiang, Jennifer; Giaever, Guri; Nislow, Corey; Hieter, Philip

    2015-01-01

    While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human–yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach. PMID:26354769

  1. Pancreatic amylase is an environmental signal for regulation of biofilm formation and host interaction in Campylobacter jejuni.

    PubMed

    Jowiya, Waheed; Brunner, Katja; Abouelhadid, Sherif; Hussain, Haitham A; Nair, Sean P; Sadiq, Sohaib; Williams, Lisa K; Trantham, Emma K; Stephenson, Holly; Wren, Brendan W; Bajaj-Elliott, Mona; Cogan, Tristan A; Laws, Andrew P; Wade, Jim; Dorrell, Nick; Allan, Elaine

    2015-12-01

    Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism. PMID:26438798

  2. Pancreatic Amylase Is an Environmental Signal for Regulation of Biofilm Formation and Host Interaction in Campylobacter jejuni

    PubMed Central

    Jowiya, Waheed; Brunner, Katja; Abouelhadid, Sherif; Hussain, Haitham A.; Nair, Sean P.; Sadiq, Sohaib; Williams, Lisa K.; Trantham, Emma K.; Stephenson, Holly; Wren, Brendan W.; Bajaj-Elliott, Mona; Cogan, Tristan A.; Laws, Andrew P.; Wade, Jim; Dorrell, Nick

    2015-01-01

    Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism. PMID:26438798

  3. An atlas of gene expression and gene co-regulation in the human retina.

    PubMed

    Pinelli, Michele; Carissimo, Annamaria; Cutillo, Luisa; Lai, Ching-Hung; Mutarelli, Margherita; Moretti, Maria Nicoletta; Singh, Marwah Veer; Karali, Marianthi; Carrella, Diego; Pizzo, Mariateresa; Russo, Francesco; Ferrari, Stefano; Ponzin, Diego; Angelini, Claudia; Banfi, Sandro; di Bernardo, Diego

    2016-07-01

    The human retina is a specialized tissue involved in light stimulus transduction. Despite its unique biology, an accurate reference transcriptome is still missing. Here, we performed gene expression analysis (RNA-seq) of 50 retinal samples from non-visually impaired post-mortem donors. We identified novel transcripts with high confidence (Observed Transcriptome (ObsT)) and quantified the expression level of known transcripts (Reference Transcriptome (RefT)). The ObsT included 77 623 transcripts (23 960 genes) covering 137 Mb (35 Mb new transcribed genome). Most of the transcripts (92%) were multi-exonic: 81% with known isoforms, 16% with new isoforms and 3% belonging to new genes. The RefT included 13 792 genes across 94 521 known transcripts. Mitochondrial genes were among the most highly expressed, accounting for about 10% of the reads. Of all the protein-coding genes in Gencode, 65% are expressed in the retina. We exploited inter-individual variability in gene expression to infer a gene co-expression network and to identify genes specifically expressed in photoreceptor cells. We experimentally validated the photoreceptors localization of three genes in human retina that had not been previously reported. RNA-seq data and the gene co-expression network are available online (http://retina.tigem.it). PMID:27235414

  4. An atlas of gene expression and gene co-regulation in the human retina

    PubMed Central

    Pinelli, Michele; Carissimo, Annamaria; Cutillo, Luisa; Lai, Ching-Hung; Mutarelli, Margherita; Moretti, Maria Nicoletta; Singh, Marwah Veer; Karali, Marianthi; Carrella, Diego; Pizzo, Mariateresa; Russo, Francesco; Ferrari, Stefano; Ponzin, Diego; Angelini, Claudia; Banfi, Sandro; di Bernardo, Diego

    2016-01-01

    The human retina is a specialized tissue involved in light stimulus transduction. Despite its unique biology, an accurate reference transcriptome is still missing. Here, we performed gene expression analysis (RNA-seq) of 50 retinal samples from non-visually impaired post-mortem donors. We identified novel transcripts with high confidence (Observed Transcriptome (ObsT)) and quantified the expression level of known transcripts (Reference Transcriptome (RefT)). The ObsT included 77 623 transcripts (23 960 genes) covering 137 Mb (35 Mb new transcribed genome). Most of the transcripts (92%) were multi-exonic: 81% with known isoforms, 16% with new isoforms and 3% belonging to new genes. The RefT included 13 792 genes across 94 521 known transcripts. Mitochondrial genes were among the most highly expressed, accounting for about 10% of the reads. Of all the protein-coding genes in Gencode, 65% are expressed in the retina. We exploited inter-individual variability in gene expression to infer a gene co-expression network and to identify genes specifically expressed in photoreceptor cells. We experimentally validated the photoreceptors localization of three genes in human retina that had not been previously reported. RNA-seq data and the gene co-expression network are available online (http://retina.tigem.it). PMID:27235414

  5. Polyaniline-graphene based α-amylase biosensor with a linear dynamic range in excess of 6 orders of magnitude.

    PubMed

    Teixeira, Sofia Rodrigues; Lloyd, Catherine; Yao, Seydou; Andrea Salvatore Gazze; Whitaker, Iain S; Francis, Lewis; Conlan, R Steven; Azzopardi, Ernest

    2016-11-15

    α-amylase is an established marker for diagnosis of pancreatic and salivary disease, and recent research has seen a substantial expansion of its use in therapeutic and diagnostic applications for infection, cancer and wound healing. The lack of bedside monitoring devices for α-amylase detection has hitherto restricted the clinical progress of such applications. We have developed a highly sensitive α-amylase immunosensor platform, produced via in situ electropolymerization of aniline onto a screen-printed graphene support (SPE). Covalently binding an α-amylase specific antibody to a polyaniline (PANI) layer and controlling device assembly using electrochemical impedance spectroscopy (EIS), we have achieved a highly linear response against α-amylase concentration. Each stage of the assembly was characterized using a suite of high-resolution topographical, chemical and mechanical techniques. Quantitative, highly sensitive detection was demonstrated using an artificially spiked human blood plasma samples. The device has a remarkably wide limit of quantification (0.025-1000IU/L) compared to α-amylase assays in current clinical use. With potential for simple scale up to volume manufacturing though standard semiconductor production techniques and subsequently clinical application, this biosensor will enable clinical benefit through early disease detection, and better informed administration of correct therapeutic dose of drugs used to treat α-amylase related diseases. PMID:27196256

  6. Gene × Smoking Interactions on Human Brain Gene Expression: Finding Common Mechanisms in Adolescents and Adults

    ERIC Educational Resources Information Center

    Wolock, Samuel L.; Yates, Andrew; Petrill, Stephen A.; Bohland, Jason W.; Blair, Clancy; Li, Ning; Machiraju, Raghu; Huang, Kun; Bartlett, Christopher W.

    2013-01-01

    Background: Numerous studies have examined gene × environment interactions (G × E) in cognitive and behavioral domains. However, these studies have been limited in that they have not been able to directly assess differential patterns of gene expression in the human brain. Here, we assessed G × E interactions using two publically available datasets…

  7. Cloning and sequencing of the gene for human. beta. -casein

    SciTech Connect

    Loennerdal, B.; Bergstroem, S.; Andersson, Y.; Hialmarsson, K.; Sundgyist, A.; Hernell, O. )

    1990-02-26

    Human {beta}-casein is a major protein in human milk. This protein is part of the casein micelle and has been suggested to have several physiological functions in the newborn. Since there is limited information on {beta}casein and the factors that affect its concentration in human milk, the authors have isolated and sequenced the gene for this protein. A human mammary gland cDNA library (Clontech) in gt 11 was screened by plaque hy-hybridization using a 42-mer synthetic {sup 32}p-labelled oligo-nucleotide. Positive clones were identified and isolated, DNA was prepared and the gene isolated by cleavage with EcoR1. Following subcloning (PUC18), restriction mapping and Southern blotting, DNA for sequencing was prepared. The gene was sequenced by the dideoxy method. Human {beta}-casein has 212 amino acids and the amino acid sequence deducted from the nucleotide sequence is to 91% identical to the published sequence for human {beta}-casein show a high degree of conservation at the leader peptide and the highly phosphorylated sequences, but also deletions and divergence at several positions. These results provide insight into the structure of the human {beta}-casein gene and will facilitate studies on factors affecting its expression.

  8. Differentiation of alpha-amylase from various sources: an approach using selective inhibitors.

    PubMed

    Quarino, L; Hess, J; Shenouda, M; Ristenbatt, R R; Gold, J; Shaler, R C

    1993-01-01

    A radial diffusion assay in an agarose/starch gel utilizing crude kidney bean extract and a commercially prepared alpha-amylase inhibitor isolated from wheat seeds was developed and assessed to determine its ability to differentiate alpha-amylase from various sources. Kidney bean extract was found to have a greater inhibitory effect on AMY2, while the wheat lectin inhibitor was found to have a greater inhibitory effect on AMY1. Neither inhibitor was found to have any effect on commercially prepared bacterial alpha-amylase extract in both liquid preparations and dried stains. Mixtures of varying concentrations of pancreatic and salivary extracts also gave interpretable results. Additionally, dried stains prepared from human body fluids having high levels of AMY2 were differentiated from dried stains prepared from human body fluids containing high levels of AMY1. PMID:8360608

  9. Gene Expression and Genetic Variation in Human Atria

    PubMed Central

    Lin, Honghuang; Dolmatova, Elena V.; Morley, Michael P.; Lunetta, Kathryn L.; McManus, David D.; Magnani, Jared W.; Margulies, Kenneth B.; Hakonarson, Hakon; del Monte, Federica; Benjamin, Emelia J.; Cappola, Thomas P.; Ellinor, Patrick T.

    2013-01-01

    Background The human left and right atria have different susceptibilities to develop atrial fibrillation (AF). However, the molecular events related to structural and functional changes that enhance AF susceptibility are still poorly understood. Objective To characterize gene expression and genetic variation in human atria. Methods We studied the gene expression profiles and genetic variations in 53 left atrial and 52 right atrial tissue samples collected from the Myocardial Applied Genomics Network (MAGNet) repository. The tissues were collected from heart failure patients undergoing transplantation and from unused organ donor hearts with normal ventricular function. Gene expression was profiled using the Affymetrix GeneChip Human Genome U133A Array. Genetic variation was profiled using the Affymetrix Genome-Wide Human SNP Array 6.0. Results We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right atrial tissues. Conclusion We found a distinct transcriptional profile between the right and left atrium, and extensive cis-associations between atrial transcripts and common genetic variants. Our results implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF. PMID:24177373

  10. The d4 gene family in the human genome

    SciTech Connect

    Chestkov, A.V.; Baka, I.D.; Kost, M.V.

    1996-08-15

    The d4 domain, a novel zinc finger-like structural motif, was first revealed in the rat neuro-d4 protein. Here we demonstrate that the d4 domain is conserved in evolution and that three related genes form a d4 family in the human genome. The human neuro-d4 is very similar to rat neuro-d4 at both the amino acid and the nucleotide levels. Moreover, the same splice variants have been detected among rat and human neuro-d4 transcripts. This gene has been localized on chromosome 19, and two other genes, members of the d4 family isolated by screening of the human genomic library at low stringency, have been mapped to chromosomes 11 and 14. The gene on chromosome 11 is the homolog of the ubiquitously expressed mouse gene ubi-d4/requiem, which is required for cell death after deprivation of trophic factors. A gene with a conserved d4 domain has been found in the genome of the nematode Caenorhabditis elegans. The conservation of d4 proteins from nematodes to vertebrates suggests that they have a general importance, but a diversity of d4 proteins expressed in vertebrate nervous systems suggests that some family members have special functions. 11 refs., 2 figs.