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Biochemical Genetics (v.46, #5-6)

Oligonucleotide Microarray Analysis of Genes Regulating Apoptosis in Chronically Ischemic and Postinfarction Myocardium by Józefa Dąbek; Aleksander Owczarek; Zbigniew Gąsior; Rafal Ulczok; Mariusz Skowerski; Andrzej Kułach; Urszula Mazurek; Andrzej Bochenek (pp. 241-247).

The pathology of cardiomyocyte death during and after myocardial infarction involves both necrosis and apoptosis. Although both mechanisms lead to cell death, participation of apoptosis in this process carries the potential of developing therapies influencing at least part of the population of dying cells. Therefore the aim of this study was to determine (using oligonucleotide microarrays) expression profiles of apoptosis-regulating genes in postinfarction myocardium, comparing chronically ischemic and healthy heart muscle. Tissue samples were obtained during elective surgery from the right cardiac auricles of three patients. The expression of 141 genes involved in fibrosis was assessed using the Affymetrix HG_U133A microarray. The patients’ transcriptomes were compared using hierarchical clusterization. Differentiating genes were determined using regression analysis and Bland–Altman graph analysis. Hierarchical clusterization demonstrated that the profile of gene expression in postinfarction myocardium was different from that in the remaining specimens. Further statistical analysis showed two important differentiating genes: FOXO3A (underexpressed in post-MI sample) and CFLAR (overexpressed in post-MI sample). The expression of apoptosis-regulating genes is significantly different in post-MI myocardium from chronically ischemic and a nonischemic myocardium. Our results suggest that CFLAR is important in the induction of apoptosis in postinfarction cardiac tissue.

Keywords: Oligonucleotide microarray; Apoptosis regulation; Myocardial infarction

Microsatellite Analysis of Genetic Variation and Population Genetic Differentiation in Autotetraploid and Diploid Rice by Li Luan; Xing Wang; Wen-Bo Long; Yu-Hua Liu; Sheng-Bin Tu; Zhan-Peng Zhao; Fan-Lun Kong; Mao-Qun Yu (pp. 248-266).

Genetic diversity and population genetic structure of autotetraploid and diploid populations of rice collected from Chengdu Institute of Biology, Chinese Academy of Sciences, were studied based on 36 microsatellite loci. Among 50 varieties, a moderate to high level of genetic diversity was observed at the population level, with the number of alleles per locus (A _e) ranging from 2 to 6 (mean 3.028) and polymorphism information content ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (H _e) varied from 0.04 to 0.76 (mean 0.370) and Shannon’s index (I) from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed slightly higher levels of A _e, H _e, and I than the diploid populations. Rare alleles were observed at most of the simple sequence repeat loci in one or more of the 50 accessions, and a core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed genetic variability mainly among autotetraploid populations rather than diploid populations (F _st = 0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and an autotetraploid and its original diploid indica male sterile lines. Group II contained only the original IR accessions. Group III was more diverse than either Group II or Group IV, comprising both autotetraploid and diploid indica restoring lines. Group IV included a japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice were somewhat dissimilar, as variation exists that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some important agricultural characters that were not found in the original diploid rice varieties.

Keywords: Autotetraploid; Diploid rice; Microsatellite (SSR); Genetic variation; Population

Cloning, Structure, and Expression Pattern of the P-450 Aromatase Gene in Rice Field Eel (Monopterus albus) by Ju-Hua Yu; Yong-Kai Tang; Jian-Lin Li (pp. 267-280).

We report the cloning, tissue expression, and structural analysis of the aromatase gene in the rice field eel (Monopterus albus). The ovary-derived cDNA (1,802 bp) has a 49 bp 5′-untranslated region (UTR), a 202 bp 3′-UTR, and a 1,551 bp open-reading frame, which encodes a protein of 517 amino acid residues with a predicted molecular weight of 58.2 kDa. The amino acid sequence alignment suggests that the rice field eel ovarian P-450 aromatase shares 63–80% identity with that of other fish species, reduced to 59–60% with brain-derived aromatases of other fishes and to 50% with human placenta aromatases. Between the 5′ and 3′ untranslated terminal regions, the rice field eel CYP19 gene contained seven introns at the same sites as in medaka and human but lacked an intron between the I-helix and the aromatase-specific conserved region. All introns conformed to the GT/AG rule. Sequence analysis of the 1,065 bp upstream of the translation start site revealed that the transcription initiation site was 51 bp upstream from the translation start site. This region had one estrogen receptor recognition half site (nt −62), five copies of an SRY/iSRY binding motif, a C/EBP (CCAAT enhancer binding protein) binding site (nt −751), chicken ovalbumin upstream promoter-transcription factor (nt −986) and GATA-2 (nt −186, −249) recognition sequences, but no binding sequence for steroidogenic factor-1 and the cAMP response element binding protein activating transcription factor family. In females, levels of relative expression were, in descending order, hypothalamus, pituitary, forebrain, ovary, and liver. In males, P450arom was detected only in the pituitary and the liver, with half the expression found in females. In fry, the P450arom expression level increased during development and was significantly higher in the brain than in the gonad.

Keywords: Monopterus albus ; P-450 aromatase; Gene structure; Regulatory sequence; Gene expression

Relationships and Hybridization among Smilax china and Its Affinities: Evidence from Allozyme Data by Aili Wang; Yeye Chen; Guangchun Chen; Joongku Lee; Chengxin Fu (pp. 281-292).

Smilax china L. is a widespread species in China with different ploidy levels. It is morphologically similar to S. davidiana, S. trinervula, and S. glauco-china. In this study, the chromosome number and the variation in allozyme patterns of eight enzyme systems with 25 alleles in 11 populations of S. china and three affinitive species were investigated. The allozyme data, together with morphological and cytological data, suggest that S. glauco-china is not closely related to the other taxa investigated. The diploid species S. davidiana and S. trinervula are involved as ancestor species and share great introgressions with S. china. In S. china, populations from Guilin and Guiyang are allotetraploid; their diploid progenitors probably are diploid populations of S. china and S. trinervula. The results suggest this species arose from multiple origins.

Keywords: Allozyme; Relationship; Polyploidy; Speciation; Smilax china

Adaptive Evolution and Frequent Gene Conversion in the Brain Expressed X-Linked Gene Family in Mammals by Liqing Zhang (pp. 293-311).

This work examines the molecular evolution of a brain-expressed X-linked gene family in the mammalian genomes of human, chimp, macaque, mouse, rat, dog, and cow. The gene structures are well conserved across family members and among the mammals in that all five members have three exons with the first two exons untranslated. Furthermore, the five members are arranged tandemly on chromosome X with Bex5, Bex1, Bex2 on the negative strand and Bex4, Bex3 on the positive strand, and this physical arrangement remains conserved among species. Sequence analyses indicate that gene conversion has been frequent and ongoing among Bex1-4, occurring in multiple species independently. All gene conversions in different species between Bex1 and Bex4, and between Bex2 and Bex3, appear to be limited to the upstream regions of the third exon, whereas the gene conversions occurred independently in different species between Bex1 and Bex2 and cover only the third exon. Bex5 appears to have little exchange of genetic information with other members, possibly due to its distance from other members. The GC content decreases from 5′-UTR, intron 1, intron 2, coding region, to 3′-UTR, reflecting faithfully the frequency of gene conversion in different regions of the Bex genes. Sequence analyses also suggest that both relaxed selective constraint and positive selection have acted on the Bex members after duplication. In particular, Bex3 shows strong evidence of positive selection and seems to have evolved a new gene function after gene duplication.

Keywords: Gene duplication; Relaxed selective constraint; Tandem arrayed genes; Positive selection; 5′-UTR; 3′-UTR; Intron

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. 312-322).

Much attention has been focused on the study of lactoferrin due to its function in antibacterial, antiviral, antifungal, anti-inflammatory, anti-oxidant, and immunomodulatory activities. A total of 60 lactoferrin (LF) gene sequences with the complete coding regions (CDS) and corresponding amino acids belonging to 11 species were analyzed, and the differentiation within and among the species was also studied. The results showed that most of the species have the stop codon TAA, with the variation of TGA for Mus musculus. The length of the LF gene with the complete CDS varies greatly, from 2,055 to 2,190 bp, due to deletion, insertion, and stop codon mutation resulting in elongation. Observed genetic diversity was higher among species than within species, and Sus scrofa had more polymorphisms than any other species. Novel amino acid variation sites were detected within several species (8 in Homo sapiens, 6 in Mus musculus, 6 in Capra hircus, 10 in Bos taurus, and 20 in Sus scrofa), which might be used to illustrate the functional variation. Differentiation of the LF gene was obvious among species, and the clustering result was consistent with the taxonomy in the National Center for Biotechnology Information.

Keywords: LF gene; Variation; Species

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. 312-322).

Keywords: LF gene; Variation; Species

Microsatellite Isolation and Characterization in Japanese Persimmon (Diospyros kaki) by Da-Long Guo; Zheng-Rong Luo (pp. 323-328).

A Biogeographic View of Apodemus in Asia and Europe Inferred From Nuclear and Mitochondrial Gene Sequences by Hitoshi Suzuki; Maria Grazia Filippucci; Galina N. Chelomina; Jun J. Sato; Keiko Serizawa; Eviatar Nevo (pp. 329-346).

Sequences of the mitochondrial cyt b gene and nuclear IRBP, RAGI, I7, and vWF genes were used to assess the evolutionary history of major lineages of Apodemus, in particular to better understand dispersal between Asia and Europe. Our data show eight extant lineages of Late Tertiary origin: Apodemus agrarius, A. semotus, A. peninsulae, A. speciosus, A. argenteus, A. gurkha, A. mystacinus, and A. sylvaticus. Monophyly of two European lineages (A. mystacinus and A. sylvaticus) and four Asian lineages (A. agrarius, A. semotus, A. peninsulae, and A. speciosus) was confirmed with high bootstrap support. Together with literature data, the available molecular data depict three crucial evolutionary events: (1) initial wide dispersal and subsequent radiation around 6 million years ago, (2) region-specific radiations in Europe and southern China around 2 million years ago, and (3) westward dispersal of A. agrarius to Europe in the Late Quaternary.

Keywords: Apodemus ; Biogeography; Late Tertiary; Molecular phylogeny; Wood mice

Mapping, Expression, and Association Study of the Bovine PSMC1 Gene by H. Guo; W.-S. Liu; A. Takasuga; K. Eyer; E. Landrito; S.-Z. Xu; X. Gao; H.-Y. Ren (pp. 347-355).

The 26S ATP-dependent protease is composed of a 20S catalytic proteasome and two PA700 regulatory modules; it plays a central role in many regulatory pathways, such as cell cycle regulation, differentiation, and apoptosis. The PA700 complex is composed of multiple subunits, including at least six related ATPases and approximately 15 non-ATPase polypeptides. PSMC1 (proteasome 26S subunit, ATPase, 1) is one of these ATPases. In this study, we amplified a fragment of 507 bp from intron 9 of the bovine PSMC1 gene and found a SNP (G/A) at position 216 in the PCR fragment. Genotyping of 138 animals from four beef breeds revealed that the average frequency for allele A (G-base) was 0.4271 (0.3269–0.5517); for allele B (A-base) it was 0.5729 (0.4483–0.6731). This SNP is significantly associated with average daily feed intake (P < 0.01), average daily gain, finishing average daily gain, body length, ratio of feed to meat, backfat thickness, and loin-muscle area (P < 0.05). Our experimental data showed that animals with an AA genotype have a significantly lower food intake, grow faster, are longer in the body, and have less backfat and bigger loin muscle; hence, their ratio of feed to meat is significantly lower. We believe that the PSMC1 SNP is a potential candidate marker for marker-assisted selection in these traits. We also found that the bovine PSMC1 gene was expressed mainly in lung, testis, and spleen. In addition, we mapped the bovine PSMC1 gene on BTA10 by an RH mapping method.

Keywords: Cattle; PSMC1 ; Mapping; Different expression; Trait association analysis

T allele for VEGF-460 Gene Polymorphism at 5′-Untranslated Region is Associated with Higher Susceptibility of Leiomyoma by Yao-Yuan Hsieh; Chi-Chen Chang; Fuu-Jen Tsai; Cheng-Chieh Lin; Chang-Hai Tsai (pp. 356-361).

Vascular endothelial growth factor (VEGF) is a regulator of angiogenesis and a mediator of sex steroid-induced cell growth and differentiation. We aimed to investigate if VEGF gene 5′-UTR −460 polymorphism could be used as markers of susceptibility in leiomyoma. Women were divided into two groups: (1) leiomyoma (n=159); (2) nonleiomyoma groups (n=131). VEGF gene −460 polymorphism were detected by polymerase chain reaction and BstUI restriction enzyme analysis. Genotypes and allelic frequencies between both groups were compared. We noted that the proportions of different VEGF polymorphisms in both groups were significantly different. Proportions of cuttable (C) homozygote/heterozygote/uncuttable (T) homozygote for VEGF in both groups were: (1) 0/32/68% and (2) 0/63/37%. Higher percentage of T homozygote and T allele presented in the leiomyoma population. Proportions of C/T alleles in both groups were: (1) 16/84% and (2) 32/68%. We concluded that T homozygotes and T allele of VEGF gene −460 polymorphism are associated with higher risk of leiomyoma development. Heterozygotes and C allele are related with lower risk of leiomyoma formation. VEGF gene polymorphism likely contributes to the pathogenesis of leiomyoma.

Keywords: Leiomyoma; Polymorphism; Vascular endothelial growth factor (VEGF)

Novel SNPs of the mtDNA ND5 Gene and Their Associations with Several Growth Traits in the Nanyang Cattle Breed by Bao Zhang; Hong Chen; Liushuai Hua; Chunlei Zhang; Xiangtao Kang; Xinzhuang Wang; Chuanying Pan; Xianyong Lan; Chuzhao Lei (pp. 362-368).

Polymorphism of the mtDNA ND5 gene was detected by PCR-SSCP and DNA sequencing methods in 714 individuals from six Chinese cattle breeds. The results showed that at the P2 locus, two kinds of haplotypes, named A and B, with three SNPs (T12900C, A12923T, C12924T) were detected. The frequencies of haplotype A in six breeds were 0.041–0.167. Polymorphism of the ND5 gene was shown to be associated with growth in the Nanyang breed. Individuals with haplotype B had greater hucklebone width than those with haplotype A when 6 months old (P < 0.01), as well as better body height, body length, body weight, and average daily gain at 6 months (P < 0.05). Therefore, haplotype B of the ND5 gene was likely to have a positive effect on growth traits at 6 months in the Nanyang breed.

Keywords: Cattle; mtDNA; ND5 gene; Polymorphism; Single nucleotide polymorphism (SNP); Growth traits

Molecular Cloning, Mapping, and Polymorphism of the Porcine SCG2 gene by Hong-Li Du; Jing Chen; Yu-Shan Zhang; Xi-Quan Zhang (pp. 369-379).

The secretogranin II (SCG2) gene is associated with the synthesis and secretion of follicle-stimulating hormone and luteinizing hormone. In the present study, we have determined the complete cDNA sequence of pig SCG2, which was submitted to GenBank with accession no. AY870646. Its complete open reading frame of 1,851 nucleotides encodes 616 amino acids. The predicted protein shares 80–87% identity with mouse, human, and bovine SCG2 proteins, and all four species share almost complete identity in the secretoneurin and EM66 domains. Pig SCG2 is a protein of 589 amino acids and 68,132 Da, preceded by a signal peptide of 27 residues. It contains nine pairs of dibasic residues, which are used as potential cleavage sites for generation of physiologically active peptides. Analysis of the SCG2 gene across the INRA-Minnesota porcine radiation hybrid panel indicates close linkage with microsatellite marker SW2608, located on Sus scrofa chromosome 15 (SSC15) q25, which harbors several QTL for ovulation rate and meat quality. Comparative sequencing and EST analysis revealed nine SNPs in porcine SCG2 cDNA, including seven SNPs in the coding region and two SNPs in the 3′ UTR. Four nonsynonymous SNPs (G622A, G1671T, C1718T, and A1790C) resulted in amino acid substitutions of Ala→Thr, Glu→Asp, Pro→Leu, and Asn→Thr, respectively.

Keywords: Porcine; SCG2 gene; Cloning; Mapping; Polymorphism

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Biochemical Genetics (v.46, #5-6)

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Biochemical Genetics (v.46, #5-6)