Check out our New Publishers' Select for Free Articles

Biochemical Genetics (v.50, #7-8)

All-Trans Retinoic Acid Induces Chromatin Remodeling at the Promoter of the Mouse Liver, Bone, and Kidney Alkaline Phosphatase Gene in C3H10T 1/2 Cells by Yang Wan; Songhai Yang; Fenyong Sun; Jiayi Wang; Qiongyu Chen; An Hong (pp. 495-507).

The alkaline phosphatase (ALP) gene is an important marker of osteoblast differentiation and bone formation. Although the molecular mechanisms of increased ALP expression in response to all-trans retinoic acid (ATRA) have been reported, the role of ATRA in chromatin structure changes remains unknown. Our results show that the expression of mouse liver, bone, and kidney ALP (mL/B/K-ALP) induced by ATRA in C3H10T 1/2 cells was related to the retinoic acid nuclear receptors, RARα and RARβ, which are not involved in the MAPK pathway. DNase I hypersensitivity analysis revealed an inducible hypersensitive site in the mL/B/K-ALP promoter at ~520 bp upstream of the transcription start site. Chromatin immunoprecipitation experiments showed a cascade of transcription cofactor recruitment events during ATRA-induced upregulation of mL/B/K-ALP. Together, our results provide a link between ATRA-induced mL/B/K-ALP gene transcription and chromatin remodeling.

Keywords: Osteoblast differentiation; Bone formation; Gene regulation; Histone modifications; MAPK pathway

Sequence Polymorphism Characteristics in the See2β Gene from Maize Key Inbred Lines and Derived Lines in China by Junjie Zhang; Min He; Yinghong Liu; Hanmen Liu; Bin Wei; Yongbin Wang; Yubi Huang (pp. 508-519).

Research on the sequence polymorphism characteristics of key genes is important in the early identification of maize inbred lines. The See2β gene functions in remobilizing leaf nitrogen and exporting it to developing grain during foliar senescence. We analyzed See2β sequences from 49 inbred lines representing four key lines and their derivatives: Huangzaosi, Mo17, Dan340, and Ye478. We found that the See2β gene had one insertion and two deletions in most Huangzaosi lines and three insertions in the Mo17 group; that the Huangzaosi, Dan340, and Ye478 lines, but not the Mo17 line, had unique indels; and that the See2β gene in lines derived from the same key inbred line had higher sequence homology, according to phenetic analysis of the inbred lines derived from Huangzaosi and Mo17. Thus, a candidate inbred line could be preliminarily identified using markers closely linked to the See2β gene combined with sequence alignment of See2β.

Keywords: Derived lines; Maize inbred lines; Sequence polymorphism; See2β

Abnormal Segregation of Alleles and Haplotypes at the Polymorphic Site of the PRNP Gene Within Promoter and Intron 1 Regions in Polish Holstein–Friesian Cattle by Janusz Strychalski; Urszula Czarnik; Tadeusz Zabolewicz (pp. 520-528).

Allele and haplotype segregation at the polymorphic sites within the promoter (23indel) and intron 1 (12indel) regions of the PRNP gene was analyzed in Polish Holstein–Friesian cattle. More 23del/del homozygotes and fewer 23ins/ins homozygotes than expected were observed in the offspring of ♂ 23ins/del × ♀ 23ins/del parents. In the offspring of ♂ 23ins/del × ♀ 23del/del parents and ♂ 23del/del × ♀ 23ins/del parents, a trend toward more 23del/del animals and fewer 23ins/del animals than expected was noted. At the 12indel polymorphic site, the only trend found was one toward fewer 12ins/ins genotypes and more 12ins/del and 12del/del genotypes than expected in the offspring of ♂ 12ins/del × ♀ 12ins/del parents. An analysis of haplotype segregation revealed more 23del-12del/23del-12del diplotypes and fewer 23ins-12ins/23ins-12ins diplotypes at the significance threshold than expected in the offspring of ♂ 23ins-12ins/23del-12del × ♀ 23ins-12ins/23del-12del parents.

Keywords: Allele and haplotype segregation; Indel polymorphisms; Holstein–Friesian cattle; PRNP gene

Down-Regulation of Pm4CL1 Gene Expression in E. coli Model by Antisense mRNA by Ha Van Huan; Yong Wang; Siliang Zhang (pp. 529-537).

The coumarate:coenzyme A ligase 1 gene (4CL1) was isolated from secondary developing xylem tissues of Pinus massoniana Lamb (Pm4CL1). The Pm4CL1 gene was inserted into a pQE30 expression vector in both sense and antisense orientations to form a pQE30-4CL1-anti4CL1. The expression level of the Pm4CL1 gene in Escherichia coli cells was inhibited by the antisense mRNA of the Pm4CL1 gene. The results of reverse transcriptase polymerase chain reaction and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the average mRNA and protein expression levels of the Pm4CL1 gene in E. coli cells were decreased by 43 and 46%, respectively. These results are an important basis for further investigation.

Keywords: Antisense mRNA; 4CL1 ; Down-regulation; Expression; E. coli

Molecular Characteristics of the Porcine TIMD4 Gene and Its Association Analysis by Jing Yuan; Zhong-Lin Tang; Shulin Yang; Ji-Yue Cao; Kui Li (pp. 538-548).

As a member of the T cell immunoglobulin domain and mucin domain (TIM) gene family, TIMD4 plays an important role in the immune response. To understand its function more precisely, we isolated it and analyzed its subcellular localization, expression pattern, and associations. The porcine TIMD4 gene included nine exons and eight introns with an open reading frame of 1086 bp encoding 361 amino acids. It had relatively high levels in liver, lymph, and spleen. The fusion protein was localized mainly in the cytoplasm of pig kidney cells (PK15). The promoter region contained a TATA box and GATA3 consensus sites. A single nucleotide polymorphism was identified in intron 3 of the porcine TIMD4 gene, and analysis indicated that it had significant associations with the 17-day red blood cell count (p = 0.0106), hemoglobin (p = 0.0149), and hematocrit (p = 0.0063) and with 32-day hemoglobin (p = 0.0140).

Keywords: Porcine; TIMD4 ; Expression pattern; Subcellular localization; Association analysis

An Interleukin-10 Gene Promoter Polymorphism (−592A/C) Associated with Type 2 Diabetes: A North Indian Study by Madhukar Saxena; C. G. Agrawal; Hemant Kumar Bid; Monisha Banerjee (pp. 549-559).

In this first report on the association of an IL-10 promoter polymorphism with type 2 diabetes mellitus in a North Indian population, the −592A/C SNP (rs1800872) was genotyped by PCR–RFLP and the IL-10 level measured using ELISA. Although no significant difference was observed in the genotypic frequencies (P = 0.657), diabetes patients carried a significantly higher number of A alleles at the −592 position, 25.6% (P < 0.001, odds ratio 0.887, 95% CI 0.670–1.184). Significant correlations were detected in postprandial glucose levels of CC-genotype patients and controls (P = 0.025), age and waist–hip ratio of CA patients and controls (P ≤ 0.001), and fasting glucose (P = 0.045) and low-density lipoprotein (P = 0.049) in all patients and controls. The serum IL-10 level was significantly higher in patients than in controls (P = 0.033). The polymorphism was significantly associated with disease incidence and its biochemical manifestations.

Keywords: SNP; Type 2 diabetes mellitus; Interleukin-10; North Indian population; ELISA; PCR–RFLP

Genotyping an ALAD Polymorphism with Real-Time PCR in Two Populations from the Iberian Peninsula by Ana O. Moreira; André Almeida; Solange Costa; Blanca Laffon; Julia García-Léston; Eduardo Pásaro; Josefina Méndez; João Paulo Teixeira (pp. 560-564).

Lead-induced toxicity varies among individuals partly because of genetic differences in their susceptibility to the metal’s effects. One genetic polymorphism associated with lead toxicity is a G-to-C transversion at position 177 in the coding region of delta-aminolevulinic acid dehydratase (ALAD), originating from two codominant alleles (ALAD1 and ALAD2). We examined the distribution of this single nucleotide polymorphism in two populations from the Iberian Peninsula. Genomic DNA was extracted from whole blood, and a real-time PCR assay was designed to determine ALAD polymorphic distribution. The distribution of MspI polymorphism was similar in the two populations studied, and allelic frequencies were comparable to those obtained in other studies of Caucasians. Further studies are needed to assess fully the functional significance of this polymorphism and its influence on the toxicokinetics of lead.

Keywords: Genotypic distribution; Real-time polymerase chain reaction; Delta-aminolevulinate acid dehydratase; Genetic susceptibility

A Heat Shock Protein Gene, CsHsp45.9, Involved in the Response to Diverse Stresses in Cucumber by Jianwu Li; He Zhang; Jianbin Hu; Junqing Liu; Keke Liu (pp. 565-578).

Heat shock proteins (Hsps) are a family of highly conserved proteins present in all organisms. They mediate a range of cytoprotective functions as molecular chaperones and are recently reported to regulate the immune response. Using suppression subtractive hybridization, we isolated and characterized a cucumber cDNA, designated CsHsp45.9, which encodes a putative heat shock protein of 45.9 kDa protein, containing three conserved DnaJ domains belonging to the Type I Hsp40 family. Real-time quantitative RT-PCR analysis revealed that CsHsp45.9 was significantly induced in cucumber leaves inoculated with downy mildew (Pseudoperonospora cubensis) in this incompatible interaction. Gene expression was also strongly up-regulated by various abiotic stresses. CsHsp45.9 was mainly expressed in flowers with a flower-specific, stamen- and pistil-predominant expression pattern. This suggests that CsHsp45.9 harbors broad-spectrum responses to both biotic and abiotic stresses and may play a role in downy mildew resistance in cucumber.

Keywords: Heat shock protein; Downy mildew; Hypersensitive response; Cucumis sativus L.

Improved RAPD-PCR for Discriminating Breeds of Water Buffalo by Rubigilda C. Paraguison; Maria Pia P. Faylon; Ester B. Flores; Libertado C. Cruz (pp. 579-584).

Isolation of Metallothionein Genes and in silico Structural Characterization of Their Proteins Using Molecular Modeling from Yak (Bos grunniens) by Liping Zhang; Jianping Wu; Xinrong Wang; Bin Liu; Binyin Ma (pp. 585-599).

Yak metallothioneins (BgMTs) are cysteine-rich metal-chelating proteins with highly conserved cysteine residues in their amino acid sequences. The 3D structures of the Cd₇-BgMTs reconstructed by molecular modeling included two domains: the β-domain with M₃(S_cys)₉ metal–thiolate clusters and the α-domain with M₄(S_cys)₁₁ metal–thiolate clusters. An unusual variant was found at position 30 (Cys30→Ser30) in BgMT-III, which is usually conserved in the mammalian MT-I/-II (Cys29) and MT-III (Cys30). The variant residue of BgMT-III may play a key role in yak genetic evolution, metal-binding activity, dynamic conformation, and heavy metal metabolism. BgMT-III contained a Thr insertion at position 5 (T⁵), which may loosen the structure of the β-domain of BgMT-III, and a conserved C⁶PCP⁹ motif, which may provide an interacting surface for protein–protein interactions. There is also an acidic hexapeptide insertion (E⁵⁵GAEAE⁶⁰) that could regulate the particular interdomain interactions and lead to the conformational change in the β-domain.

Keywords: Yak (Bos grunniens); Metallothioneins (MTs); Molecular modeling; Structure function

Effect of Chitinase on Resistance to Fungal Pathogens in Sea Buckthorn, Hippophae rhamnoides, and Cloning of Class I and III Chitinase Genes by Yan-Lin Sun; Soon-Kwan Hong (pp. 600-615).

Sea buckthorn (Hippophae rhamnoides L.) is naturally distributed from Asia to Europe. It has been widely planted as an ornamental shrub and is rich in nutritional and medicinal compounds. Fungal pathogens that cause diseases such as dried-shrink disease are threats to the production of this plant. In this study, we isolated the dried-shrink disease pathogen from bark and total chitinase protein from leaves of infected plants. The results of the Oxford Cup experiment suggested that chitinase protein inhibited the growth of this pathogen. To improve pathogen resistance, we cloned chitinase Class I and III genes in H. rhamnoides, designated Hrchi1 and Hrchi3. The full-length cDNA of the open reading frame region of Hrchi1 contained 903 bp encoding 300 amino acids and Hrchi3 contained 894 bp encoding 297 amino acids. Active domain analysis, protein types, and secondary and 3D structures were predicted using online software.

Keywords: Hippophae rhamnoides ; Chitinase protein; Chitinase genes; Cloning; Pathogen resistance

Genetic Changes Following Hybridization and Genome Doubling in Synthetic Brassica napus by Yanhao Xu; Hong Xu; Xiaoming Wu; Xiaoping Fang; Jianbo Wang (pp. 616-624).

Genetic changes were investigated in two sets of independently synthesized Brasscia napus allopolyploids by the AFLP approach in the present study. We found that 1.17 % of the loci showed genetic changes following both hybridization and genome doubling in the synthesized B. napus F04J2 relative to its diploid progenitors, B. rapa (AA genome) and B. oleracea (CC genome). No significant difference between the proportion of A-genome-specific genetic changes and that of C-genome-specific genetic changes was detected in B. napus F04J2. Approximately 0.6 % of the loci displayed genetic changes following somatic genome doubling in the amphidiploid B. napus DCE11 relative to the amphihaploid in the dimorphic plants. This study showed that rapid genetic changes occurred after hybridization and/or genome doubling in synthesized B. napus allopolyploids and indicated that both hybridization and genome doubling could affect the genomic architecture in newly formed allopolyploids.

Keywords: Hybridization; Genome doubling; AFLP analysis; Brassica napus

Expression and Characterization of a Functional Single-Chain Variable Fragment (scFv) Protein Recognizing MCF7 Breast Cancer Cells in E. coli Cytoplasm by Ilham Omer Mahgoub (pp. 625-641).

Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production.

Keywords: Single-chain fragment; Origami DE3; Bacterial cytoplasm expression; Protein solubility; Breast cancer cells; Immunofluorescence; Flow cytometry analysis

Assessment of Genetic Diversity in Zingiberaceae Through Nucleotide Binding Site-Based Motif-Directed Profiling by Raj Kumar Joshi; Sujata Mohanty; Basudeba Kar; Sanghamitra Nayak (pp. 642-656).

Functional motif-directed profiling was performed with 15 nucleotide binding site (NBS) primer–enzyme combinations to identify and elucidate the phylogenetic relationships among 15 genotypes of the family Zingiberaceae. We retrieved 167 polymorphic bands (24.85 %), with an average of 11.13 bands per primer. Mean polymorphism rates were detected using MseI (26 %), RsaI (21 %), and AluI (28 %) as restriction enzymes. The polymorphism information content (PIC) for each NBS primer–enzyme combination ranged from 0.48 to 0.76 with a mean value of 0.65. The 38 NBS profiling markers had PIC values ranging from 0.3 to 0.6 and exhibited good power to discriminate between genotypes. Comparison of NBS profiling with microsatellite data for the same set of genotypes exhibited a correlation value of 0.78, P ≤ 0.001. Our study suggests that genetic variability assessment could be more efficient if it targeted genes that exhibit functionally relevant variation, rather than random markers.

Keywords: Nucleotide binding site; Genetic diversity; NBS profiling; Zingiberaceae

Sections

Personal tools

Biochemical Genetics (v.50, #7-8)

Sections

Personal tools

Local resources

Biochemical Genetics (v.50, #7-8)