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Biochemical Genetics (v.50, #9-10)
Polymorphisms of Intron 1 and the Promoter Region at the PRNP Gene in BSE-Free Caracu Cattle by Cleber Eduardo Galvão; Grácia Maria Soares Rosinha; Cristiane Camargo Sanches; Carina Elisei; Flábio Ribeiro Araújo; Gelson Luís Dias Feijó; Roberto Augusto de Almeida Torres Jr.; Cleber Oliveira Soares (pp. 657-669).
The infectious prion protein PrPSc is encoded by the PRNP gene. In cattle, insertion/deletion (indel) polymorphisms are among the changes that occur in this gene, the most studied of which are within intron 1 (12 bp) and the promoter region (23 bp). Sequence variants in this gene may affect the formation of PrPSc. In the present study, nucleotide variability in specific regions of the PRNP gene in Caracu cattle free of bovine spongiform encephalopathy was investigated to determine the genotypic profile of each animal within the group. Caracu cattle exhibited high allele frequency for the two polymorphic regions studied, 12ins (70 %) and 23ins (72.5 %), genotype frequencies of 50 % for 12ins/ins and 50 % for 23ins/del, and a high frequency of the 12ins–23ins haplotype (57.5 %). Of the 40 animals sampled, 15 had the 12ins–23ins/12ins–23ins diplotype.
Keywords: Bovine spongiform encephalopathy; PRNP ; Prion; Indel; Caracu cattle
PICcalc: An Online Program to Calculate Polymorphic Information Content for Molecular Genetic Studies
by Sándor Nagy; Péter Poczai; István Cernák; Ahmad Mousapour Gorji; Géza Hegedűs; János Taller (pp. 670-672).
Molecular Cloning and Characterization of the 5′-Flanking Regulatory Region of the Carbonic Anhydrase Nacrein Gene of the Pearl Oyster Pinctada fucata and Its Expression by Tomoyuki Miyashita; Akiko Takami; Ryosuke Takagi (pp. 673-683).
The carbonic anhydrase nacrein participates in the formation of the nacreous or prismatic layer of Pinctada fucata. We isolated a genomic clone containing the nacrein gene and cloned the 5′-flanking region. Within the 1336 bp 5′ flanking region, we identified putative cis-acting elements, including the TATA box (TATAAAA) at −82 bp, and AP1 (−819 bp) and Oct-1 (−1244 bp) binding sites. In addition to the mantle, the nacrein gene is also expressed in the adductor muscle, liver, and foot. These results showed that nacrein not only takes part in the formation of the hard tissue but also might be involved in acid–base balance, ion transport, and maintenance of ionic concentration. In vitro transcription experiments showed that the addition of human c-jun activates transcription from the nacrein promoter. This is the first report of a promoter from a gene that controls the formation of the hard tissue of mollusk shells.
Keywords: Carbonic anhydrase; Nacrein; Pinctada fucata ; Biomineralization; Promoter; C-jun
Genotype Frequencies of Drug-Metabolizing Enzymes Responsible for Purine and Pyrimidine Antagonists in a Healthy Asian-Indian Population by Sandhya N. Iyer; Ashwini V. Tilak; Manjari S. Mukherjee; Rekha S. Singhal (pp. 684-693).
Purine and pyrimidine antimetabolites are used to treat leukemias, autoimmune diseases, and solid tumors. Detection of slow metabolizers before administration of the drugs is necessary to prevent any subsequent drug toxicity. With this aim, we determined the frequencies of normal and slow alleles in our population. Polymorphisms in genes encoding cytidine deaminase (CDA), dihydropyrimidine dehydrogenase (DPYD), and thiopurine-S-methyltransferase (TPMT) were documented in 225 healthy volunteers. The polymorphisms typed included CDA*3, DPYD*2A, TPMT*2A, TPMT*3B, and TPMT*3C. Methods used for genotyping included standard PCR–RFLP and allele-specific PCR reactions. The frequencies were 0.44 % for DPYD*2A, 0.67 % for TPMT*3B, and 0.89 % for TPMT*3C. The CDA*3 and TPMT*2A alleles were not detected. Although these polymorphisms have been demonstrated to be associated with drug toxicity in other populations, they appear to be very rare in the adult Indian population.
Keywords: Asian Indians; Thiopurine-S-methyltransferase; Cytidine deaminase; Dihydropyrimidine dehydrogenase; Genotyping; India; Pharmacogenomics
Molecular Characterization of Prolactin cDNA and Its Expression Pattern in Skin Tissue of Liaoning Cashmere Goat
by W. L. Bai; R. H. Yin; W. Q. Jiang; G. B. Luo; R. L. Yin; C. Li; Z. H. Zhao (pp. 694-701).
Identification of a Toll-Like Receptor 1 in Guinea Fowl (Agelastes niger) by Yanhua Wu; Wenke Ruan; Defeng Cui; Huanrong Li (pp. 702-716).
Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, thus playing important roles in host defense. This study determined the first sequence of a TLR1 type 1 in the guinea fowl (GFTLR1). The open reading frame of GFTLR1 type 1 contains 2,115 nucleotides and encodes 705 amino acids. Amino acid analysis indicated that GFTLR1 type 1 shares 92.3 % homology with the green jungle fowl, 92.1 % with the chicken, 90.4 % with the turkey, and 84.4 % with Cooper’s hawk. Genetic patterns were identified within the TLR1 type 1 of the chicken and the guinea fowl. GFTLR1 type 1 was found to have 92 polymorphic amino acid sites, of which 16 were in the leucine-rich repeat (LRR) domain, 3 in a C-terminal LRR domain, and 6 in a Toll/interleukin-1 receptor domain. The data showed that avian TLR1 type 1 genes are under purifying selection and highly conserved, because dN/dS was less than 1.
Keywords: Chicken; Guinea fowl; Polymorphism; Toll-like receptor (TLR)
The Transferability of Illumina Canine BeadChip Single-Nucleotide Polymorphisms (SNPs) to American Mink (Neovison vison)
by Peter Foged Larsen; Ditte Demontis; Vivi Hunnicke Nielsen; Janne Pia Thirstrup; Volker Loeschcke; Cino Pertoldi (pp. 717-721).
Characterization of a Chitinase (Chit62) from Serratia marcescens B4A and Its Efficacy as a Bioshield Against Plant Fungal Pathogens by S. Babashpour; S. Aminzadeh; N. Farrokhi; A. Karkhane; K. Haghbeen (pp. 722-735).
Chitinases have been suggested to be involved in pathogen–antagonist interaction during biological control progress of plant pathogenic fungi. Here, a recombinant bacterial chitinase originally from Serratia marcescens B4A was produced, purified, and assayed biochemically to ascertain the activity and determine the kinetics parameters. Active enzyme was used to determine its biocontrol features against fungal phytopathogens. The results demonstrated that the optimum pH and temperature for the enzyme activity were 6.0 and 55 °C, respectively. The K m and V max values were 3.30 mg ml−1 and 0.92 units, respectively. The recombinant chitinase was demonstrated to be highly active in controlling fungal pathogens.
Keywords: Heterologous expression; Protein activity assay; Chitinase; Fungal biocontrol
A Bioinformatics Approach to Characterize Mammalian Selenoprotein T by Mohamed E. Moustafa; Hussien A. Antar (pp. 736-747).
The exact function and trafficking of selenoprotein T (SelT) are still unclear. This study was focused on using bioinformatics analysis as an approach to understanding the structure–function relationship of SelT and the trafficking of SelT between cellular compartments. Blast analysis revealed that SelT is present in mammals, birds, frogs, zebra fish, and green algae. Structural analyses revealed that SelT contains a CxxU motif in a thioredoxin-like fold, suggesting a redox function of SelT. Cysteine (Cys) residues were found in the place of selenocysteine in SelT Cys homologs in insects, roundworms, and plants. The SignalP program recognized signal peptides in both SelT and SelT Cys homologs. Mammalian SelT was predicted to contain an N-terminal signal peptide of 19 amino acid residues, which may be involved in targeting SelT to the endoplasmic reticulum. Finally, SelT may be localized in the plasma membrane in addition to its presence in the Golgi apparatus and the endoplasmic reticulum.
Keywords: Calreticulin/calnexin cycle; CxxU motif; SelT Cys homologs; Thioredoxin-like fold; Trafficking
Diversity of Apis mellifera Subspecies from Turkey Revealed by Sequence Analysis of Mitochondrial 16s rDNA Region by Fulya Özdil; Fatma Ilhan (pp. 748-760).
Mitochondrial DNA sequence variation can be used to infer honeybee evolutionary relationships. In this study, DNA sequence diversity of the mitochondrial 16s rDNA region was investigated in 112 honeybees from 15 populations in Turkey, which is mainly populated with Apis mellifera anatoliaca, A. m. caucasica, and A. m. meda. The study revealed 11 haplotypes for this segment, with 13 variable sites and nine parsimony informative sites. The haplotypes were not discriminated according to their geographical locations in a neighbor-joining dendrogram based on 16s rDNA sequences available in Genbank, but all the haplotypes obtained in this study are clustered with published haplotypes such as A. mellifera TAS (AF214666) and A. m. ligustica (EF116868) and with some unpublished Genbank records (HQ318928, HQ318934, and HQ318938). This study expands the knowledge of the mitochondrial 16s rDNA region, and it presents the first comprehensive sequence analysis of this region in Turkish honeybees.
Keywords: Apis mellifera L.; 16s rDNA; Turkey; DNA sequence diversity
A Versatile Agrobacterium-Mediated Transient Gene Expression System for Herbaceous Plants and Trees by Lei Zheng; Guifeng Liu; Xiangnan Meng; Yanbang Li; Yucheng Wang (pp. 761-769).
Plant transient expression is a powerful method used widely for the functional characterization of genes and protein production. In comparison with stable transformation, it has the advantages of being simple, quick, economical, and effective. In the present study, we developed a novel transient gene expression system based on Agrobacterium-mediated transformation. This system is simple and convenient and allows for high transient expression levels. Hyperosmotic pretreatment of plants significantly improved the transient expression in this system. Furthermore, other factors, including acetosyringone concentration, cocultivation time, and Agrobacterium cell density, significantly influenced transient expression efficiency. The results showed that this method is suitable for use with herbaceous plants (such as tobacco and Arabidopsis) and trees (such as birch, poplar, tamarisk, cork, willow, and aralia), suggesting that it may be applied widely in plant transient expression studies.
Keywords: Agrobacterium tumefaciens ; Plants; GUS staining; Transformation; Transient gene expression
Population Structure and Genetic Diversity in Popular Rice Varieties of India as Evidenced from SSR Analysis by Priti Upadhyay; C. N. Neeraja; C. Kole; Vikas Kumar Singh (pp. 770-783).
We report here on the phylogenetic analysis, population substructure, and identification of molecular tags of 25 popular rice varieties and four landraces from different ecological belts of India employing a set of 52 simple sequence repeat (SSR) markers. Genetic analysis using the SSR markers categorized the genotypes into two major clusters, distributed according to their pedigree. Population structure analysis suggested that the optimum number of subpopulations was three (K = 3) in the popular varieties and landraces. At K = 5 the allelic distribution was much more similar to the phylogenetic dendrogram. The molecular diversity and population structure analysis indicated that there is not much variation among the popular rice cultivars of India. The study has identified SSR markers producing unique alleles, which should aid in the precise identification, maintenance, and genetic purity analysis of rice varieties.
Keywords: Rice; Population structure; SSR; Molecular tags; Genetic diversity
A 45-bp Insertion/Deletion Polymorphism in Uncoupling Protein 2 is Not Associated with Obesity in a Chinese Population by Xiaoqiu Liu; Bin Zhang; Xin Liu; Yue Shen; Jiana Li; Nan Zhao; Lina Ma; Qingmao Du; Lisheng Liu; Jingbo Zhao; Xingyu Wang (pp. 784-796).
The association of a 45-bp insertion/deletion (UCP2-45 bp I/D) polymorphism in uncoupling protein 2 with body mass index (BMI) remains controversial. A case–control study was conducted to examine the association in a Chinese population. The 1,526 subjects recruited in downtown Beijing and genotyped included 616 obese subjects with BMI >28 and 910 age- and gender-matched controls with BMI <24. The association of the polymorphisms with obesity was estimated using multivariate logistic regression in three models of inheritance. The odds ratios were 1.08 (95 % CI 0.846–1.368; P = 0.551) in the dominant model, 0.931 (0.751–1.154; P = 0.513) in the additive model, and 1.18 (0.550–2.550; P = 0.666) in the recessive model. The overall comparison of the genotype distributions in obese and control subjects using the chi-square test yielded P = 0.801. Our study demonstrated no association between UCP2-45 bp I/D and BMI variation in the Chinese population.
Keywords: UCP2; Polymorphism; Obese; Chinese population; Case–control study
Impact of Geographic Range on Genetic and Chemical Diversity of Indian Valerian (Valeriana jatamansi) from Northwestern Himalaya by V. Sundaresan; G. Sahni; R. S. Verma; R. C. Padalia; S. Mehrotra; Sanjog T. Thul (pp. 797-808).
An effort was made to determine the impact of geographic range on genetic richness and chemical constituents of Valeriana jatamansi Jones, an herb indigenous to the northwestern Himalaya. The genetic structure of 16 accessions from two major divisions of Uttarakhand state (Kumaon and Garhwal) was analyzed by ISSR markers. Overall genetic diversity among the populations was 45 %, with a cumulative range of 35–92 % similarity for most of the high-altitude plants and a comparatively narrow range, 50–88 %, for the population below the altitude of 1,800 m. Likewise, a remarkable predictability was evident from the chemical constituents on an individual basis. In principal component analysis, most of the accessions fall into two major groups and are classified as chemotypes based on the percentage of similar chemical constituents; these are mostly correlated to altitude. Geographic distance seems to influence the genetic and chemical variability, indicating the genetic inbreeding within the population.
Keywords: Terpenoid profiling; Patchouli alcohol; Principal component analysis; ISSR markers; Genetic cluster analysis
Gene Expression Profiles of HLA-G1 Overexpressed in hES Cells by Yajing Zhu; Sanjun Zhao; Hongxi Zhao; Yuanqing Yao (pp. 809-821).
The goals of this study were to analyze the change in the global gene expression profile of exogenous human leukocyte antigen-G1 (HLA-G1) overexpressed in human embryonic stem (hES) cells and to explore the molecular mechanism by which the overexpression of HLA-G1 modifies immunologic pathways. Microarray and quantitative real-time PCR analyses were performed to quantify the differential expression pattern of HLA-G1 + H1 hES cells. The results showed that HLA-G1 differentially regulated the expression of 425 genes with at least a twofold increase or decrease. These differentially expressed genes were classified into 13 functional groups, including cellular components, biological processes, and molecular functions. The pathways of focal adhesion, the TGF-β signaling pathway, and the immune response were the most predominantly affected. The synergism of these genes could explain the mechanism of the immunosuppression of HLA-G1 + H1 hES cells. Thus, the expression pattern reflected a broad spectrum of roles of HLA-G1 in hES cells.
Keywords: Human embryonic stem cell; Human leukocyte antigen G1; Microarray