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Biochemical Genetics (v.50, #11-12)

A Combination of Structural and Cis-Regulatory Factors Drives Biochemical Differences in Drosophila melanogaster Malic Enzyme by Teresa Z. Rzezniczak; Thomas E. Lum; Robert Harniman; Thomas J. S. Merritt (pp. 823-837).

The evolutionary significance of molecular variation is still contentious, with much current interest focusing on the relative contribution of structural changes in proteins versus regulatory variation in gene expression. We present a population genetic and biochemical study of molecular variation at the malic enzyme locus (Men) in Drosophila melanogaster. Two amino acid polymorphisms appear to affect substrate-binding kinetics, while only one appears to affect thermal stability. Interestingly, we find that enzyme activity differences previously assigned to one of the polymorphisms may, instead, be a function of linked regulatory differences. These results suggest that both regulatory and structural changes contribute to differences in protein function. Our examination of the Men coding sequences reveals no evidence for selection acting on the polymorphisms, but earlier work on this enzyme indicates that the biochemical variation observed has physiological repercussions and therefore could potentially be under natural selection.

Keywords: Malic enzyme (Men); Molecular evolution; Genetic variation; Enzyme function

Selection of an Effective Small Interference RNA to Silence Myostatin Gene Expression in Sheep Fibroblast Cells by Jian Lu; Dan Sun; Lingyang Xu; Guobin Lu; Fuping Zhao; Caihong Wei; Li Zhang; Jiatong Ding; Bichun Li; Lixin Du (pp. 838-847).

Myostatin (MSTN), a member of the TGF-β superfamily, has been identified as a negative regulator of skeletal muscle mass. Inactivating mutations in the MSTN gene are responsible for the development of a hypermuscular phenotype. The aim of this study was to identify an effective small interfering RNA (siRNA) to knockdown the myostatin gene in sheep fibroblast cells. Four siRNAs targeting sheep myostatin were synthesized and tested. Quantitative RT-PCR showed that siRNA1, siRNA2, siRNA3, and siRNA5 significantly reduced myostatin transcript levels by 72, 68, 56, and 76 % (P < 0.05), respectively. Western blot analysis showed that myostatin protein expression was significantly reduced by 76 % using siRNA1 and by 65 % using siRNA5 (P < 0.05). Therefore, siRNA1 and siRNA5 may have the potential to knockdown myostatin gene expression and increase sheep meat production, which should be a focus of future studies.

Keywords: RNA interference; Small interfering RNA; Myostatin; Sheep fibroblasts

Characterization of Soil Bacterial Communities in Rhizospheric and Nonrhizospheric Soil of Panax ginseng by Yi Xin Ying; Wan Long Ding; Yong Li (pp. 848-859).

A culture-independent approach was used to evaluate the bacterial community in rhizospheric and nonrhizospheric soil in which Panax ginseng had grown for 3 years. For each sample, soil was randomly collected from multiple sampling points and mixed thoroughly before genomic DNA extraction. Universal primers 27f and 1492r were used to amplify 16S rRNA genes. Clone libraries were constructed using the amplified 16S rRNA genes, and 192 white clones were chosen for further sequencing. After digestion with restriction endonuclease, 44 operational taxonomic units (OTUs) were generated for rhizospheric and 21 OTUs for nonrhizospheric soils, and the clones of each OTU were sequenced. Blast analysis showed that bacillus, acidobacteria, and proteobacteria were the dominant populations in rhizospheric soil, and proteobacteria were dominant in nonrhizospheric soil. Phylogenetic results showed that bacillus and acidobacteria were clustered into the group of uncultured bacteria in rhizospheric soil; however, proteobacteria were the unique dominant in nonrhizospheric soil.

Keywords: Panax ginseng ; Rhizospheric soil; Bacterial community; ARDRA

XPD Gene rs13181 Polymorphism and DNA Damage in Human Lymphocytes by Marta Wlodarczyk; Grazyna Nowicka (pp. 860-870).

Genetic heterogeneity influencing enzyme activity may change the capacity to repair DNA damage induced by environmental and endogenous factors. This study aims to assess the impact of Lys751Gln (A/C) polymorphism in the XPD gene, encoding an enzyme involved in the nucleotide excision repair pathway, on individual DNA damage. The DNA damage in human lymphocytes (% DNA in the tail) was quantified by means of single-cell gel electrophoresis. Baseline levels of DNA damage significantly differ between AA homozygotes and carriers of the C allele, and the observed differences were not related to age, gender, or smoking status. It seems that the AA variant is associated with enhanced protection against oxidative DNA damage.

Keywords: XPD ; Genetic polymorphism; Comet assay; DNA damage

Frequency Distribution of Q188R, N314D, Duarte 1, and Duarte 2 GALT Variant Alleles in an Indian Galactosemia Population by Ramandeep Singh; Babu R. Thapa; Gurjit Kaur; Rajendra Prasad (pp. 871-880).

Classical galactosemia is a genetic disorder caused by mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. The Q188R and N314D mutations are the most frequently cited GALT gene mutations. N314D is further associated with two variants, Duarte 1 and Duarte 2. Nevertheless, no reports are available on the clinical and molecular spectrum of galactosemia from the Indian population. The present study was designed to establish the frequency of these two most common mutations and their variants in Indian galactosemia patients so as to determine a single most common mutation/polymorphism for establishing the DNA-based diagnosis of galactosemia. Three alleles were found to be present at a frequency of 0.036 (Q188R), 0.40 (N314D), and 0.39 (D2); no D1 alleles were found. A significantly higher frequency of the Duarte 2 allele in our population suggests the presence of a milder form of galactosemia, which can be well managed by early diagnosis and dietary management.

Keywords: Allele; Duarte; GALT; Mutations; Polymorphisms

A Simple and Efficient DNA Isolation Method for Salvia officinalis by Jelena M. Aleksić; Danilo Stojanović; Bojana Banović; Radiša Jančić (pp. 881-892).

We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4 M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6 μg DNA g⁻¹ of dry leaf tissue, and the absorbance ratios 260/280 and 260/230 nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.

Keywords: Salvia officinalis ; DNA isolation; CTAB; PVP 10; Activated charcoal; NaCl; Sevag; PCR; 26S rDNA; rps16-trnK

MTHFR C677T Polymorphism Among Three Mendelian Populations: A Study from North India by Seerat Sachdeva; Kallur N. Saraswathy; Mahima Gulabani; Shweta Kaushik; M. P. Sachdeva; Manju Puri; S. L. Malik (pp. 893-897).

An SNP in the MyoD1 Gene Intron 2 Associated with Growth and Carcass Traits in Three Duck Populations by Y. Wu; J. S. Pi; A. L. Pan; Y. J. Pu; J. P. Du; J. Shen; Z. H. Liang; J. R. Zhang (pp. 898-907).

Myogenic differentiation 1 (MyoD1) genes belong to the MyoD gene family and play key roles in growth and muscle development. This study was designed to investigate the effects of variants in the MyoD1 gene on duck growth and carcass traits. Three duck populations (Cherry Valley, Jingjiang, and Muscovy) were sampled, their growth and carcass traits were measured, and they were genotyped using the PCR–RFLP method. The results showed one novel polymorphism, an alteration in intron 2 of the MyoD1 gene (A to T). It was associated with the traits of weight at 8 weeks, carcass weight, breast muscle weight, leg muscle weight, eviscerated percentage, percentage of leg muscle weight, dressing percentage, and lean meat percentage. This alteration in intron 2 of MyoD1 may be linked with potential major loci or genes affecting some growth and carcass traits.

Keywords: Duck (Anas platyrhynchos); MyoD1 ; SNP; PCR–RFLP

Comparative Transcriptomic Profiling of Two Tomato Lines with Different Ascorbate Content in the Fruit by Antonio Di Matteo; Adriana Sacco; Rosalba De Stefano; Luigi Frusciante; Amalia Barone (pp. 908-921).

In recent years, interest in tomato breeding for enhanced antioxidant content has increased as medical research has pointed to human health benefits from antioxidant dietary intake. Ascorbate is one of the major antioxidants present in tomato, and little is known about mechanisms governing ascorbate pool size in this fruit. In order to provide further insights into genetic mechanisms controlling ascorbate biosynthesis and accumulation in tomato, we investigated the fruit transcriptome profile of the Solanum pennellii introgression line 10-1 that exhibits a lower fruit ascorbate level than its cultivated parental genotype. Our results showed that this reduced ascorbate level is associated with an increased antioxidant demand arising from an accelerated oxidative metabolism mainly involving mitochondria, peroxisomes, and cytoplasm. Candidate genes for controlling ascorbate level in tomato fruit were identified, highlighting the role of glycolysis, glyoxylate metabolism, and purine breakdown in modulating the ascorbate pool size.

Keywords: Antioxidants; Berry quality; Carbohydrate metabolism; Solanum spp.; Reactive oxygen species

Effects of Selenium and Exendin-4 on Glucagon-Like Peptide-1 Receptor, IRS-1, and Raf-1 in the Liver of Diabetic Rats by Ghinwa M. Barakat; Mohamed E. Moustafa; Anwar B. Bikhazi (pp. 922-935).

Selenium and exendin-4 exert antidiabetic effects by unknown mechanisms. Herein, we investigated their effects on the expression of glucagon-like peptide-1 receptor (GLP-1R), insulin receptor substrate-1 (IRS-1), and Raf-1 in the livers of rats with streptozotocin-induced diabetes. Diabetic rats were injected intraperitoneally with exendin-4 (0.03 μg/kg body weight) twice daily or treated with 5 ppm selenium as sodium selenite in drinking water for 4 weeks. Both selenium and exendin-4 reduced the hyperglycemia in diabetic rats. Induction of diabetes mellitus resulted in decreased level of GLP-1R and increased levels of IRS-1 and Raf-1 in the liver. Treatment of diabetic rats with selenium or exendin-4 resulted in increased level of GLP-1R and decreased levels of IRS-1 and Raf-1 in the liver, compared with the levels in diabetic rats. Therefore, the antidiabetic actions of selenium and exendin-4 involve their effects on GLP-1R, IRS-1, and Raf-1 levels in the liver.

Keywords: Antidiabetic; Diabetes mellitus; Exendin-4; GLP-1 receptor; Glutathione peroxidase-1

Molecular Cloning and Biochemical Analysis of Tyrosinase from the Crested Ibis in China by Jing Yang; Xiaolin Liu; Jianqin Zhang; Baoping Qing; Baozhong Lu (pp. 936-945).

The crested ibis, one of the most endangered birds in the world, could benefit from research into its genetic diversity as a tool for conservation in the future. Tyrosinase is thought to play a major role in the production of common yellow to black melanins in birds. We have cloned and sequenced four exons of the crested ibis tyrosinase gene and discovered that the amino acid sequence has high similarity to zebra finch tyrosinase (93 %), followed by chicken (91 %) and quail (91 %). Some functional and structural domains in the crested ibis tyrosinase coding area were found to be conserved during evolution. Nine sequence variants were found in the partial coding sequence, one in exon 1 and eight in exon 4. Sequence variant 1 (SV1) shows intermediate polymorphism (0.25 < PIC < 0.5), and further study is needed to determine whether it can be used as a potential molecular marker in crested ibis artificial breeding programs.

Keywords: Crested ibis; Molecular cloning; Biochemical analysis; Tyrosinase

Molecular Characterization and Expression Analysis of VSIG4 from the Asian Yellow Pond Turtle, Mauremys mutica by Mi Zhao; Yan Shi; Xinping Zhu; Kunci Chen; Debo Pan; Chengqing Wei (pp. 946-958).

V-set and immunoglobulin domain-containing protein 4 (VSIG4), a member of the immunoglobulin superfamily, plays an important role in the immune system. This study isolated and characterized a cDNA encoding VSIG4 (MaVSIG4) from the Asian yellow pond turtle (Mauremys mutica). The MaVSIG4 cDNA is 1840 bp long and contains an open reading frame of 1,182 bp that encodes a polypeptide of 372 amino acids. The genomic sequence of MaVSIG4 spans 7,682 bp, with six exons and five introns. The phylogenetic tree shows that MaVSIG4 is most closely related to Gallus gallus VSIG4. The expression analysis by real-time PCR reveals that MaVSIG4 is ubiquitously expressed in various healthy tissues, with a higher expression level in the liver. After immune stimulation, the expression level of MaVSIG4 sharply decreased in the liver, heart, and kidney at 12 h (P < 0.01). These results provide a basis for further study of the function of MaVSIG4 in the turtle’s immune system.

Keywords: Asian yellow pond turtle; Mauremys mutica ; Serratia marcescens ; V-set and immunoglobulin domain-containing protein 4 (VSIG4); Immune responses

Polyphyletic Origin of the Genus Amaurornis Inferred from Molecular Phylogenetic Analysis of Rails by Luzhang Ruan; Yushi Wang; Jinrun Hu; Yi Ouyang (pp. 959-966).

Comprehensive Analysis of UGT1A1 Genetic Polymorphisms in Chinese Tibetan and Han Populations by Xiaoqing Zhang; Xiaohong Meng; Yuewen Wang; Wei Yan; Jin Yang (pp. 967-977).

The study of polymorphism of the UGT1A1 gene has not been reported in the Chinese Tibetan population, and there are no comparisons of genetic polymorphism in the gene between Chinese Han and Tibetan populations. In this study, we directly sequenced the functional regions of the UGT1A1 gene in 200 unrelated healthy Chinese volunteers, detecting 20 variations (including five novel ones). The distributions of allele and genotype frequencies differ between the two groups. UGT1A1*6 is the major reduced functional variant in the populations, and the *27 allele was detected only in the Han group. Differences in the frequencies of the UGT1A1*6/*63 genotype between the Tibetan and Han populations were statistically significant (P = 0.009). Our genetic data might provide fundamental information for the advance of personalized medicine and will facilitate genotype-phenotype studies in larger populations.

Keywords: Chinese; Genetic polymorphisms; Haplotype; UGT1A1 gene

Phylogenetic Genomewide Comparisons of the Pentatricopeptide Repeat Gene Family in indica and japonica Rice by Shaobo Li; Qingping Sun; Minghua Hu; Shaoqing Li; Youlin Zhu; Yingguo Zhu (pp. 978-989).

More than 400 pentatricopeptide repeat (PPR) genes have been found in higher plants, but most of them have not been functionally analyzed and their origins are still obscure. In this study, we performed phylogenetic genomewide comparisons of the PPR gene family in indica and japonica rice to explore the expansion mechanisms of these genes in higher plants. The functions of PPR genes in plant CMS/Rf systems are also discussed. The results indicate that (1) unequal crossing over participated in the expansion of the newly evolved PPR genes in indica and japonica rice genomes, (2) CMS/Rf systems are different in monocots and dicots, (3) the BT-type CMS/Rf system exists in both indica and japonica rice, and (4) both the PPR gene family and the BT-type CMS/Rf system may have existed before the divergence of indica and japonica rice.

Keywords: indica rice; japonica rice; Pentatricopeptide repeat; Unequal crossing over; CMS/Rf system

Decreasing Activity and Altered Protein Processing of Human Iduronate-2-sulfatase Mutations Demonstrated by Expression in COS7 Cells by Ratana Charoenwattanasatien; James R. Ketudat Cairns; Siriporn Keeratichamroen; Phannee Sawangareetrakul; Pranoot Tanpaiboon; Duangrurdee Wattanasirichaigoon; Suthipong Pangkanon; Jisnuson Svasti; Voraratt Champattanachai (pp. 990-997).

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Biochemical Genetics (v.50, #11-12)