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Biochemical Genetics (v.47, #11-12)
Molecular Characterization and Functional Commonality of Nucleophosmin/Nucleoplasmin in Two Cyprinid Fish by Nan Wu; Chang-Jian Li; Jian-Fang Gui (pp. 749-762).
Nucleophosmin/nucleoplasmin has been studied mostly in mammals and amphibians. To clarify the characteristics and function of nucleophosmin/nucleoplasmin in teleost fish, we cloned a full-length cDNA sequence from two cyprinid fish, Carassius auratus gibelio and Carassius auratus. Molecular characterization and multiple sequence alignments suggested that they are the homologs of nucleophosmin. RT-PCR and Western blot detected a specific expression in gonads, and immunofluorescence localization revealed their distribution in oogenic and spermatogenic cells. Furthermore, a sperm decondensation function was demonstrated by immunodepletion and in vitro sperm decondensation experiments. The data suggest that the cloned nucleophosmin should share expressional and functional characterization with nucleoplasmin and therefore provide novel evidence for a functional commonality of nucleophosmin and nucleoplasmin in fish.
Keywords: Oogenesis; Spermatogenesis; Sperm decondensation; Carassius auratus gibelio ; Carassius auratus
Molecular Characterization, Mapping, and Haplotype Analysis of Porcine Matrix Metalloproteinase Genes MMP1 and MMP10 by Xiao Wu; Yuchun Pan (pp. 763-774).
Matrix metalloproteinases (MMPs) are a family of enzymes that cleave protein components of the extracellular matrix, such as collagens, laminin, fibronectin, and proteoglycans, playing a role in degradation of the matrix of the uterus and in embryo implantation. We report the identification of two members of the MMP gene family in swine. The porcine MMP1 and MMP10 genes comprise 10 exons and 9 introns spanning approximately 8,460 and 7,030 bp. Of 28 potential single nucleotide polymorphisms found in the genomic region, five polymorphic positions were analyzed using PCR-RFLP. Allelic frequencies and haplotypes were analyzed in five pig breeds (n = 280). The AC haplotype of MMP1 and ATG haplotype of MMP10 were not detected in two foreign pig breeds. Association analysis in a French Large White population (n = 164, total four traits) showed no association between haplotypes and reproduction performance. In addition, porcine MMP1 and MMP10 were mapped on SSC9p13 and SSC2q21, respectively, in agreement with comparative mapping data.
Keywords: Pig; MMP1; MMP10; SNP; Mapping
Two Different Size Classes of 5S rDNA Units Coexisting in the Same Tandem Array in the Razor Clam Ensis macha: Is This Region Suitable for Phylogeographic Studies? by Juan Fernández-Tajes; Josefina Méndez (pp. 775-788).
For a study of 5S ribosomal genes (rDNA) in the razor clam Ensis macha, the 5S rDNA region was amplified and sequenced. Two variants, so-called type I or short repeat (~430 bp) and type II or long repeat (~735 bp), appeared to be the main components of the 5S rDNA of this species. Their spacers differed markedly, both in length and nucleotide composition. The organization of the two variants was investigated by amplifying the genomic DNA with primers based on the sequence of the type I and type II spacers. PCR amplification products with primers EMLbF and EMSbR showed that the long and short repeats are associated within the same tandem array, suggesting an intermixed arrangement of both spacers. Nevertheless, amplifications carried out with inverse primers EMSinvF/R and EMLinvF/R revealed that some short and long repeats are contiguous in the same tandem array. This is the first report of the coexistence of two variable spacers in the same tandem array in bivalve mollusks.
Keywords: 5S rDNA; Nontranscribed spacer variations; Cluster organization; Razor clam; Ensis macha
Contrary Effects of BMP-2 and ATRA on Adipogenesis in Mouse Mesenchymal Fibroblasts by Fenyong Sun; Qiuhui Pan; Jiayi Wang; Shanyin Liu; Zhi Li; Yongchun Yu (pp. 789-801).
This study evaluates the effects of bone morphogenetic protein 2 (BMP-2) and all trans retinoic acid (ATRA) on adipogenesis in primary mouse embryo fibroblasts (MEFs). In BMP-2-treated MEFs, lipid accumulation and substantial induction of the adipocyte specific marker 442-aP2 suggested the conversion of MEFs into adipocytes. Such adipogenesis was found to be mediated through sequential induction of C/EBPα, C/EBPβ, and PPARγ. Both the BMP/Smad and BMP/p38 pathways contributed to the adipocyte differentiation. Contrary to the effects of BMP-2, ATRA was demonstrated to inhibit adipocyte differentiation in MEFs. Semi-quantitative RT-PCR analysis revealed that ATRA caused a selective inhibition of both the basal and induction levels of C/EBPα and PPARγ, without altering the expression pattern of C/EBPβ. Taken together, these data suggest the roles of BMP-2 and ATRA in adipogenic differentiation of primary MEFs, and the possible molecular mechanism that involves the regulation of C/EBPα, C/EBPβ, and PPARγ.
Keywords: Bone morphogenetic protein; All trans retinoic acid; Adipogenesis; Mouse mesenchymal fibroblasts
Expression Pattern and Prokaryotic Expression for Peking Duck Insulin-Like Growth Factor-I in Escherichia coli by Yan Wu; Xiao-lin Liu; Hong-wei Xiao; Jie Wang; Fei Jiang; Sheng Zhao; Yu Liu (pp. 802-811).
Insulin-like growth factor-I (IGF-I) is an important regulator of growth, development, and metabolism in animals. This study investigated its expression pattern in various tissues in Peking duck and in vivo. The result showed that the recombinant protein of IGF-I was expressed mainly in the form of inclusion bodies in the Escherichia coli cell (37.4 kD). Western blotting indicated that the recombinant protein had the antigenicity of IGF-I. Reverse-transcript PCR and real-time fluorescent quantitative PCR analyses showed that IGF-I was expressed in the heart, liver, abdominal fat, breast muscle, spleen, lung, kidney, gizzard, and small intestine of 8-week-old birds. In the RT-PCR results, IGF-I was expressed highest in the heart and lung and lowest in abdominal fat and breast muscle in male and female birds. In the real-time fluorescent quantitative PCR results, IGF-I was expressed highest in the gizzard and lowest in the lung in all ducks.
Keywords: Expression pattern; Insulin-like growth factor-I (IGF-I); Peking duck (Anas platyrhynchos); Prokaryotic expression
An Exonic Insertion Encodes an Alanine Stretch in Porcine Synapsin I
by Claus Hedegaard; Emøke Bendixen; Poul Henning Jensen; Christian Bendixen; Knud Larsen (pp. 812-816).
Organization of a β and α Globin Gene Set in the Teleost Atlantic Cod, Gadus morhua by Katrín Halldórsdóttir; Einar Árnason (pp. 817-830).
Developmental globin gene expression and gene switching in vertebrates have been extensively studied. Globin gene regions have been characterized in some fish species and show linked α and β loci. Understanding coordinated expression between α and β globin genes in fish is of importance for further insights into globin gene regulation in teleosts and higher vertebrates. We characterize linked β and α globin genes in Atlantic cod, pulled from the Atlantic cod genome with a PCR research strategy, by screening a genomic λ library and primer walking. The genes are oriented tail-to-head (5′–3′), differing from the head-to-head orientation in transcriptional polarity characteristic of teleostean globin genes. Four tandem repeats are found in an intergenic region of 1500 base pairs. One microsatellite, which consists primarily of atg tandem repeats, has an open reading frame. The globin genes and open reading frame have a CCAAT promoter element and TATA boxes. The promoters of the open reading frame and the β gene share an 89-bp block (with 100% identity) that probably regulates transcription.
Keywords: Hemoglobin; β/α globin genes; Regulatory elements; Atlantic cod; Gadus morhua
Genetic Variability and Geographic Differentiation in Thymus daenensis subsp. daenensis, an Endangered Medicinal Plant, as Revealed by Inter Simple Sequence Repeat (ISSR) Markers by Mehdi Rahimmalek; Babak Bahreininejad; Mojtaba Khorrami; Badraldin Ebrahim Sayed Tabatabaei (pp. 831-842).
Thymus daenensis is an aromatic medicinal plant endemic to Iran. We used inter simple sequence repeat (ISSR) markers to detect genetic polymorphism in this herb using 17 T. daenensis accessions collected from different geographic regions in Iran. The 15 primers chosen for analysis revealed 256 bands, of which 228 (88.9%) were polymorphic. Jaccard’s similarity indices based on ISSR profiles were subjected to UPGMA cluster analysis. The generated dendrogram revealed two major groups. The Tc group included the accessions collected from the center of the Zagros Mountains, and the Te group was collected from the extremes of the Zagros range. A principal coordinate analysis confirmed the results of clustering. The results showed that the divergence of accessions based on the Zagros Mountains is more logical in comparison with classification on the basis of provincial borders. Gene diversity and expected heterozygosity were greater in the Tc group than in the Te group, suggesting that the germplasm collected from the center of the Zagros Mountains is more variable.
Keywords: Thymus daenensis ; Threatened medicinal plant; ISSR markers; Conservation
Biochemical and Molecular Characterization of Some Centaurea Species Growing in the Eastern Anatolia Region of Turkey by Nalan Yildirim; Serap Sunar; Guleray Agar; Sedat Bozari; Ozkan Aksakal (pp. 850-859).
Random amplified polymorphic DNA (RAPD) and fatty acid (FAME) profiles were used to examine phenotypic and genetic relationships among 16 Centaurea species growing wild in the eastern Anatolia region of Turkey. Thirteen decamer primers were used to examine polymorphism. According to the RAPD results, 99 amplicons in the size range of 50–1000 bp were produced from 13 primers in 16 Centaurea species. Genetically four distinct groups were determined among the species of Centaurea, which represents high genetic variation. In the 16 species, 14 fatty acids were determined according to FAME results. Both FAME and RAPD results showed that C. virgata is genetically different from other species. The differences in the composition of fatty acids among Centaurea species suggest that fatty acid profiles could be used to differentiate among some of these species. Results of this study show that RAPD and FAME analyses are consistent.
Keywords: Centaurea species; Genetic variation; FAME; RAPD
Two Sympatric Phylogroups of the Chinese Water Deer (Hydropotes inermis) Identified by Mitochondrial DNA Control Region and Cytochrome b Gene Analyses by Hung Sun Koh; Bae Keun Lee; Jinxing Wang; Seon Wook Heo; Kyung Hee Jang (pp. 860-867).
Mitochondrial DNA (mtDNA) control region (927 bp) and cytochrome b gene (1,140 bp) sequences of the Chinese water deer (Hydropotes inermis) from China and Korea were obtained to examine the taxonomic status of two subspecies, H. i. inermis from China and H. i. argyropus from Korea. Two sympatric mtDNA clades (a major clade from China and Korea and a minor clade from Korea) with an average genetic distance of 2.1% in the control region and 1.3% in the cytochrome b gene were detected. These findings are not consistent with the current classification by pelage color. We propose a reconsideration of the validity of the subspecies designation by the statistical comparison of morphological characters including body color. The major common mtDNA phylogroup in the two allopatric subspecies could be explained by the contiguous distribution of the Chinese water deer from east China to Korea until recent years. The restriction in the range and number of the Chinese subspecies after the last glacier might have caused the disappearance of the minor phylogroup in China. The taxonomic status of the two groups in Korea should be clarified using nuclear DNA marker analyses as well as morphological characters including pelage color.
Keywords: Hydropotes inermis ; China and Korea; Sympatric phylogroups
Recurring G12S Mutation of HRAS in a Chinese Child with Costello Syndrome with High Alkaline Phosphatase Level
by Huiwen Zhang; Jun Ye; Xuefan Gu (pp. 868-872).
Noninvasive Genotyping of the Red-Legged Partridge (Alectoris rufa, Phasianidae): Semi-Nested PCR of Mitochondrial DNA from Feces by Monica Guerrini; Filippo Barbanera (pp. 873-883).
DNA-based studies using avian feces are scarce and deal only with large-sized species. The red-legged partridge, Alectoris rufa, is a medium-sized member of the order Galliformes. Our goal was to set up a fast, noninvasive procedure for mitochondrial DNA (mtDNA) genotyping of A. rufa fecal samples. We focused on the protected population from Elba Island (Tuscan Archipelago National Park, Italy). Dry A. rufa fecal samples (n = 30) were collected in winter. Both the cytochrome b gene (1,092 bp) and the control region (ca. 1,155 bp) were amplified by means of semi-nested PCRs. Twenty-five samples were successfully sequenced for both genes: 8 showed A. rufa mtDNA lineage and 17 chukar partridge (A. chukar), an exotic species. Mixed maternal ancestry suggests A. rufa × A. chukar hybridization. Our protocol allows noninvasive mtDNA genotyping of any Alectoris species and appears suitable to investigate protected populations as well as those existing either at very low density or inhabiting poorly accessible regions.
Keywords: Alectoris ; Avian feces; Noninvasive sampling; Partridges; Semi-nested PCR
Comparative Analysis of Genetic Diversity and Population Structure of Sipunculus nudus as Revealed by Mitochondrial COI Sequences by Xiaodong Du; Zian Chen; Yuewen Deng; Qingheng Wang (pp. 884-891).
Genetic diversity and population structure of Sipunculus nudus were evaluated using a 652 base pair fragment of the mitochondrial cytochrome oxidase I gene. The populations were collected from Beihai, Sanya, and Xiamen. A total of 71 polymorphic sites defined 16 distinct haplotypes. The mean haplotype diversity and nucleotide diversity of the three populations were 0.9354 ± 0.0168 and 0.0035 ± 0.0018, respectively. Analysis at the intrapopulation level showed that the Beihai population had the greatest haplotype and nucleotide diversity, followed by the Xiamen and Sanya populations. Analysis of molecular variance showed significant genetic differentiation among the three populations (Fst = 0.0796, P < 0.05). The present results revealed that S. nudus populations had a high level of genetic diversity and distinct population structures.
Keywords: Sipunculus nudus ; Genetic diversity; Population structure; COI gene
Necessity of Quantum Coherence to Account for the Spectrum of Time-Dependent Mutations Exhibited by Bacteriophage T4 by W. Grant Cooper (pp. 892-910).
Transcriptase measurements of quantum expectations due to time-dependent coherent states populating informational DNA base-pair sites, designated by G–C → *G–*C, G–C → G′–C′, and A–T → *A–*T, provide a model for transcription and replication of time-dependent DNA lesions exhibited by bacteriophage T4. Coherent states are introduced as consequences of hydrogen bond arrangement, keto-amino → enol-imine, where product protons are shared between two sets of indistinguishable electron lone-pairs and thus participate in coupled quantum oscillations at frequencies of ~1013 s−1. The transcriptase deciphers and executes genetic specificity instructions by implementing measurements on superposition proton states at *G–*C, G′–C′, and *A–*T sites in an interval Δt ≪ 10−13 s. Decohered states participate in Topal–Fresco replication, which introduces substitutions *C → T, *G → A, G′ → T, and G′ → C, but superposition *A–*T states are deleted. These results imply an evolutionary shift favoring A–T richness.
Keywords: Molecular evolution mechanism; Transcription enhancement of mutation; Transcriptase quantum processing; Coherent states in DNA; Entanglement; Replication; T4 phage genetics
Erratum to: Bioinformatics Analysis of Lactoferrin Gene for Several Species
by Jing-Fen Kang; Xiang-Long Li; Rong-Yan Zhou; Lan-Hui Li; Fu-Jun Feng; Xiu-Li Guo (pp. 911-911).