Journal of Genetics and Genomics

Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating

Zhengchao Wang, Yongqing Jiang, Lizhi Lu, Ruihua Huang, Qingchao Hou, Fangxiong Shi

2007, 34(6): 477-485. doi: 10.1016/S1673-8527(07)60052-6

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Abstract:
Cyclic nucleotide-gated ion channels (CNGs) are distributed most widely in the neuronal cell. Great progress has been made in molecular mechanisms of CNG channel gating in the recent years. Results of many experiments have indicated that the stoichiometry and assembly of CNG channels affect their property and gating. Experiments of CNG mutants and analyses of cysteine accessibilities show that cyclic nucleotide-binding domains (CNBD) bind cyclic nucleotides and subsequently conformational changes occurred followed by the concerted or cooperative conformational change of all four subunits during CNG gating. In order to provide theoretical assistances for further investigation on CNG channels, especially regarding the disease pathogenesis of ion channels, this paper reviews the latest progress on mechanisms of CNG channels, functions of subunits, processes of subunit assembly, and conformational changes of subunit regions during gating.

C-terminal 76 Amino Acids of eRF3 Are Not Required for the Binding of Release Factor eRF1a from Euplotes octocarinatus

Li Song, Yuyao Wang, Baofeng Chai, Wei Wang, Aihua Liang

2007, 34(6): 486-490. doi: 10.1016/S1673-8527(07)60053-8

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Abstract:
Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRF1-dependent GTPase. Polypeptide release factor eRF3 consists of N-terminal variable region and C-terminal conserved part. C-terminal part of eRF3 is responsible for termination of the translation. In the present study, the C-terminal of Euplotes octocarinatus eRF3 (eRF3C) and truncate eRF3C lacking 76 amino acids in C-terminal (eRF3Ct) were expressed in Escherichia coli. The recombinant GST-eRF3C and GST-eRF3Ct polypeptides were purified by affinity chromatography using glutathione Sepharose 4B column. After enzymatic cleavage of GST tail, the eRF3C and eRF3Ct protein were obtained. Pull-down analysis showed that the recombinant GST-eRF3C and GST-eRF3Ct polypeptides interacted with E. octocarinatus polypeptide chain release factor eRF1a. This result suggested that the C-terminal of eRF3 having 76 amino acids were not required for the binding of eRF1a in Euplotes octocarinatus.

An Improved Enucleation Method of Bovine Somatic Cell Nuclear Transfer

Song Hua, Zhipeng Zhang, Chi Zhang, Yong Zhang

2007, 34(6): 491-496. doi: 10.1016/S1673-8527(07)60054-X

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Abstract:
The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch²-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase ? stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls (87.4% vs. 64.4%, P < 0.05). Furthermore, centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.

Generation of Antibodies Against DMRT1 and DMRT4 of Oreochromis aurea and Analysis of Their Expression Profile in Oreochromis aurea Tissues

Jinling Cao, Zhemin Cao, Tingting Wu

2007, 34(6): 497-509. doi: 10.1016/S1673-8527(07)60055-1

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Abstract:
Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing (Doublesex/Mab-3 DNA-binding motif) gene, is highly conserved across species. Vertebrate DMRT1 (DM-related transcription factor 1) expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 (DM-related transcription factor 4) and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain, there is little similarity outside the DM-domain. To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with β-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.

Genetic Variation of Mitochondrial D-loop Region and Evolution Analysis in Some Chinese Cattle Breeds

Shangang Jia, Hong Chen, Guixiang Zhang, Zhigang Wang, Chuzhao Lei, Ru Yao, Xu Han

2007, 34(6): 510-518. doi: 10.1016/S1673-8527(07)60056-3

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Abstract:
The complete mitochondrial D-loop region from 123 individuals in 12 Chinese cattle breeds and two individuals in Germany Yellow cattle breed was sequenced and analyzed. The results were shown as follows: 93 variations and 57 haplotypes were detected, and the average number of nucleotide difference was 22.708, nucleotide diversity (d) was 0.0251 ± 0.00479, and haplotype diversity (Hd) was 0.888 ± 0.026, indicating very high genetic diversity in Chinese cattle breeds. In the Neighbor-Joining tree, 13 cattle breeds were divided into two main clades, Bos taurus and Bos indicus; new Clade ? had only one sequence from Apeijiaza cattle breed in Tibet, which was similar to that of yak at a higher level than other cattle breeds, proving the introgression of genes from the yak. The proportions of Bos taurus and Bos indicus were 64.3% and 35.7% respectively in Xigazê Humped cattle breed, and 50.0% and 50.0% respectively in Apeijiaza cattle breed, which revealed that Tibet cattle included Bos indicus haplotypes. The importance of Yunnan cattle in the origin of Chinese cattle was also confirmed based on their abundant haplotypes. Then, a very special haplotype i1 discovered in 27 Chinese cattle breeds, including 11 breeds in this study and 16 breeds in the GenBank, played the role of a nucleus in Chinese zebu and was further discussed. At the same time, the construction of Chinese zebu core group based on haplotype i1 validated the distinct origin of Bos indicus in Tibet, which was different from that of the other cattle breeds with zebu haplotypes in China.

Morphological, Anatomical and Genetic Analysis for a Rice Mutant with Abnormal Hull

Quanfang Zhang, Jiandi Xu, Yun Li, Peizhou Xu, Hongyu Zhang, Xianjun Wu

2007, 34(6): 519-526. doi: 10.1016/S1673-8527(07)60057-5

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Abstract:
A mutant with abnormal hull was first discovered from a twin-seedling strain W2555 in rice (Oryza sativa L.). The mutant had sparse branches and decreased number of florets from the base to the peak. Frequently, the florets at the top of the panicle did not develop completely. The underdeveloped florets often showed slender and white in their life cycle. Genetic analysis indicated that the mutant traits were controlled by a single recessive gene (temporarily designated asah). ah gene controlled the development of inflorescence meristem and the flower organ. The florets of mutant showed degenerated lemma and palea. Stamens and lodicules were homeoticly transformed into pistils and palea/lemma-like structures, respectively. It seemed that ah mutant phenotypes of the homeotic conversions in lodicules and stamens were very similar to that of the B loss-of-function spw1 gene reported previously in rice.

Cloning of a MADS Box Gene (GhMADS3) from Cotton and Analysis of Its Homeotic Role in Transgenic Tobacco

Yulong Guo, Qinlong Zhu, Shangyong Zheng, Mingyang Li

2007, 34(6): 527-535. doi: 10.1016/S1673-8527(07)60058-7

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A MADS box gene (GhMADS3) was cloned from cotton (Gossypium hirsutum L.) based on EST sequences. The predicted protein sequence of GhMADS3 showed 85%, 73%, and 62% identity with Theobroma cacao TcAG, Antirrhinum majus FAR, and Arabidopsis thaliana AG, respectively, and was grouped with AG homologues when the full length sequences excluding N-extensions were compared. GhMADS3 expressed in the wild type cotton flower primarily in stamens and carpels, which was comparable to AG in Arabidopsis. However, it was not expressed in floral buds of a homeotic cotton variant chv1. Ectopic expression of GhMADS3 in tobacco (Nicotiana tabacum L.) resulted in flowers with sepal-to-carpel and petal-to-stamen transformation. The carpelloid first whorl organs, with stigmatic tissue on their upper edges, had a white appearance when compared with the dark green color of the wild type sepals. At times, long filaments were observed at the fusion site of the first carpelloid oranges. The second whorl organs in staminoid were usually smaller than the wild type and the color was changed from pink to white. These results suggest that GhMADS3 has a homeotic role in flower development.

Analysis of Differentially Expressed Genes in Genic Male Sterility Cotton (Gossypium hirsutum L.) Using cDNA-AFLP

Xiaoding Ma, Chaozhu Xing, Liping Guo, Yangcang Gong, Hailin Wang, Yunlei Zhao, Jianyong Wu

2007, 34(6): 536-543. doi: 10.1016/S1673-8527(07)60059-9

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Abstract:
cDNA amplified fragment length polymorphism (cDNA-AFLP) analysis was used to investigate the differentially expressed genes between sterile and fertile plants of ms5ms6 double-recessive genic male sterility (GMS) two-type line cotton (Gossypium hirsutum L.) at different stages, i.e., sporogenous cell stage, pollen mother cell (PMC) stage, and pollen grain stage. Seventeen differentially expressed fragments were identified. Functional analysis indicated that their corresponding genes may participate in the processes of signal transduction, transcription, energy metabolism, and plant cell wall development. Northern blot demonstrated the credibility of the result of cDNA-AFLP. A sterility restorer factor-like gene, which only expressed in fertile anther and was notably homologous to T cytoplasm male sterility restorer factor 2 of maize (Zea mays L.), was identified in this research.

Comparative Assessment of Genetic Diversity of Peanut (Arachis hypogaea L.) Genotypes with Various Levels of Resistance to Bacterial Wilt Through SSR and AFLP Analyses

Huifang Jiang, Boshou Liao, Xiaoping Ren, Yong Lei, Emma Mace, Tingdong Fu, J.H. Crouch

2007, 34(6): 544-554. doi: 10.1016/S1673-8527(07)60060-5

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Abstract:
Bacterial wilt (BW) caused by Ralstonia solanacearum is an important constraint to peanut (Arachis hypogaea L.) production in several Asian and African countries, and planting BW-resistant cultivars is the most feasible method for controlling the disease. Although several BW-resistant peanut germplasm accessions have been identified, the genetic diversity among these has not been properly investigated, which has impeded efficient utilization. In this study, the genetic relationships of 31 peanut genotypes with various levels of resistance to BW were assessed based on SSR and AFLP analyses. Twenty-nine of 78 SSR primers and 32 of 126 AFLP primer combinations employed in this study were polymorphic amongst the peanut genotypes tested. The SSR primers amplified 91 polymorphic loci in total with an average of 3.14 alleles per primer, and the AFLP primers amplified 72 polymorphic loci in total with an average of 2.25 alleles per primer. Four SSR primers (14H06, 7G02, 3A8, 16C6) and one AFLP primer (P1M62) were found to be most efficient in detecting diversity. The genetic distance between pairs of genotypes ranged from 0.12 to 0.94 with an average of 0.53 in the SSR data and from 0.06 to 0.57 with an average of 0.25 in the AFLP data. The SSR-based estimates of the genetic distance were generally larger than that based on the AFLP data. The genotypes belonging to subsp.fastigiata possessed wider diversity than that of subsp. hypogaea. The clustering of genotypes based on the SSR and AFLP data were similar but the SSR clustering was more consistent with morphological classification of A. hypogaea. Optimum diverse genotypes of both subsp. hypogaea and subsp. fastigiata can be recommended based on this analysis for developing mapping populations and breeding for high yielding and resistant cultivars.

Analysis of Codon Usage Between Different Poplar Species

Meng Zhou, Chunfa Tong, Jisen Shi

2007, 34(6): 555-561. doi: 10.1016/S1673-8527(07)60061-7

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Codon usage is the selective and nonrandom use of synonymous codons to encode amino acids in genes for proteins. The analysis of codon usage may improve the understanding of codon preferences between different species and allow to rebuild the codons of exogenous genes to increase the expression efficiency of exogenous genes. Here, codon DNA sequence (CDS) of four poplar species, including Populus tremuloides Michx., P. tomentosa Carr., P. deltoides Marsh., and P. trichocarpa Torr. & Gray., is used to analyze the relative frequency of synonymous codon (RFSC). High-frequency codons are selected by high-frequency (HF) codon analysis. The results indicate that the codon usage is common for all four poplar species and the codon preference is quite similar among the four poplar species. However, CCT encoding for Pro, and ACT coding for Thr are the preferred codons in P. tremuloides and P. tomentosa, whereas CCA coding for Pro, and ACA coding for Thr are preferred in P. deltoides and P. trichocarpa. The codons such as TGC coding for Cys, TTC coding for Phe, and AAG coding for Lys, are preferred in the poplar species except P. trichocarpa. GAG coding for Glu is preferred only in P. deltoides, while the other three poplar species prefer to use GAA. The commonness of preferred codon allows exogenous gene designed by the preferred codon of one of the different poplar species to be used in other poplar species.

Analysis of Genetic Effects of Nuclear-Cytoplasmic Interaction on Quantitative Traits: Genetic Model for Diploid Plants

Lide Han, Jian Yang, Jun Zhu

2007, 34(6): 562-568. doi: 10.1016/S1673-8527(07)60062-9

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A genetic model was proposed for simultaneously analyzing genetic effects of nuclear, cytoplasm, and nuclear-cytoplasmic interaction (NCI) as well as their genotype by environment (GE) interaction for quantitative traits of diploid plants. In the model, the NCI effects were further partitioned into additive and dominance nuclear-cytoplasmic interaction components. Mixed linear model approaches were used for statistical analysis. On the basis of diallel cross designs, Monte Carlo simulations showed that the genetic model was robust for estimating variance components under several situations without specific effects. Random genetic effects were predicted by an adjusted unbiased prediction (AUP) method. Data on four quantitative traits (boll number, lint percentage, fiber length, and micronaire) in Upland cotton (Gossypium hirsutum L.) were analyzed as a worked example to show the effectiveness of the model.

2007 Vol. 34, No. 6