三种猕猴属动物Fas基因的克隆

从腹股沟淋巴结或血液中提取总RNA,根据人的Fas基因的cDNA序列设计上、下游引物,通过RT-PCR扩增出三种猕猴属动物Fas基因的cDNA片段,将片段克隆到pGEM-TEasy载体中,筛选阳性克隆并进行序列测定。恒河猴Fas基因编码序列为1005bp,GenBank收录号为AY007572;熊猴的编码序列为996bp,GenBank收录号为AF326208;短尾猴的编码序列为933bp,GenBank收录号为AF332357。对五种已知的灵长类动物的Fas基因进行序列比较,结果表明Fas基因的保守区域具有高度保守性,而处于编码序列中间的一段低复杂度序列在不同的物种间具有很高的变异性。 Abstract：Total RNA were extracted from inguinal lymph nodes o r blood.The Fas cDNA fragments of three macaca species were obtained by RT-P CR using the upper and low primers according to the Fas gene of human,and th en cloned into pGEM-T Easy vector.The positive clones were sequenced.For the fir st time we cloned the Fas genes of Macaca mulatta,Macaca assamensis and Macaca arctoides,the encoding sequences are 1005bp, 996bp and 933bp, respect ively.Finally we compared the sequences of Fas genes among five primate spec ies that have already been submitted to GenBank.The results confirmed the conser vation of regions in two ends of Fas genes,and the high variability of a low complexity region in middle of every Fas gene.     

Computational identification and evolutional analysis of Piwi subfamily in 11 Drosophila species

The complete genome sequences of 11 Drosophila species provide an opportunity to investigate the gene family evolution in closely related species. Drosophila Piwi subfamily, including three members, piwi, Aub and Ago3, has attracted increasing attention as it participates in the biogenesis of piRNA. Here, we identified 33 Piwi homologs from the genome of 11 Drosophila species. The full-length cDNA sequences ofpiwi and Aub genes were obtained by using New GENSCAN Web Server. The Ago3 homologs were difficult regarding full-length information because they had long introns. The genomic structure of Piwi subfamily genes are highly conserved among diverse Drosophila species. Insect piwi and Aub genes have long first introns. The average length of the first intron is 1 284 bp for piwi and 840 bp for Aub, which is much larger than those of other introns （93 bp for Piwi and 54 bp for Aub）. However, this phenomenon is not observed in mammalian piwi genes. We also found that there were abundant repeat sequences in both exons and introns of insect Ago3 genes. Due to recent insertions of long terminal repeat elements in four Drosophila species, part of the third introns exhibit higher conservation than adjacent exons and other introns. An evolutional tree created by Minimum Evolution method indicates that mammalian piwi genes are more closely related to the insect Ago3 Piwi subfamily.     

利用差减杂交分离具有进化保守性的睾丸表达的基因

The mechanisms of sexual development and regulation display extraordinary diversity between phyla. However, recent studies show that there must be underlying evolutionary conservatism between different mechanisms. In order to examine evolutionary conservatism in molecular mechanisms of sex determination, the natural sex reversal process of the rice field eel (Monopterus albus) was used to identify conserved functional genes involved in sex determination and differentiation. Firstly, a mini cDNA library was constructed from the cDNAs expressed specifically in the human testis. The cDNA library was screened with first strand cDNA probes from the intersex gonads of rice field eels. Secondly, likely positive clones were arrayed onto two nylon membranes, which were then hybridized with the first strand cDNAs probes from rice field eel intersex and female gonads respectively. Two positive clones were obtained, which showed obvious differential expression between intersex and female gonads. DNA sequencing analysis reveal that the insert fragments of these two clones were 895 bp and 596 bp long respectively. BLAST results indicate that the 895 bp fragment is the 3′ part of the No.AJ011779 gene, located on 6q21, encoding a homologue of yeast Sec63p protein. An interesting finding is that one member of the human Sec gene family, Sec61γ , is expressed in the testis and involved in the process of testis development. The 596 bp fragment was 97% identical with the No.BG723880 EST, located on clone RP11 12M5 of chromosome 1 This needs further study. We infer that these two genes should also play a role in sexual development.     

Comparative Analysis of Codon Usage Patterns Among Mitochondrion, Chloroplast and Nuclear Genes in Triticum aestivum L.

In many organisms, the difference in codon usage patterns among genes reflects variation in local base compositional biases and the intensity of natural selection. In this study, a comparative analysis was performed to investigate the characteristics of codon bias and factors in shaping the codon usage patterns among mitochondrion, chloroplast and nuclear genes in common wheat （Triticum aestivum L.）. GC contents in nuclear genes were higher than that in mitochondrion and chloroplast genes. The neutrality and correspondence analyses indicated that the codon usage in nuclear genes would be a result of relative strong mutational bias, while the codon usage patterns of mitochondrion and chloroplast genes were more conserved in GC content and influenced by translation level. The Parity Rule 2 （PR2） plot analysis showed that pyrimidines were used more frequently than purines at the third codon position in the three genomes. In addition, using a new alterative strategy, 11, 12, and 24 triplets were defined as preferred codons in the mitochondrion, chloroplast and nuclear genes, respectively. These findings suggested that the mitochondrion, chloroplast and nuclear genes shared particularly different features of codon usage and evolutionary constraints.     

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.     

Complete mitochondrial genome of Oxya chinensis （Orthoptera, Acridoidea）

The complete sequence of Oxya chinensis （0. chinensis） mitochondrial genome is reported here. It is 15,443 bp in length and contains 75.9% A＋T. The protein-coding genes have a similar A＋T content （75.2%）. The initiation codon of the cytochrome oxidase subunit I gene in the mitochondrial genome of O. chinensis appears to be ATC, instead of the tetranucleotides that have been reported in Locusta migratoria （L migratoria） mitochondrial genome. The sizes of the large and small ribosomal RNA genes are 1319 and 850 bp, respectively. The transfer RNA genes have been modeled and showed strong resemblance to the dipteran transfer RNAs, and all anticodons are identical to those of dipteran. The A＋T-rich region is 562 bp, shorter than that of other known Orthoptera insects. The six conserved domains were identified within the A＋T-rich region by comparing its sequence with those of other grasshoppers. The result of phylogenetic analysis based on the dataset containing 12 concatenated protein sequences confirms the close relation-ship of O. chinensis with L migratoria.     

Identification and organ expression of peroxisome proliferator activated receptors in brown trout (Salmo trutta f. fario)

Although widely studied in mammals, little information about fish peroxisome proliferator activated receptors (PPARs) is yet available. As a baseline for future studies, the three PPAR isotypes were identified in brown trout (Salmo trutta f. fario) and their organ distribution pattern was established. The cDNA fragments encoding PPARs alpha, beta and gamma were amplified by PCR, and the deduced sequences of the correspondent peptides were compared with other species sequences. Both the 183 amino acid sequence from PPARalpha and the 103 amino acid sequence from PPARbeta shared high levels of homology with the correspondent peptides of other fishes and terrestrial vertebrates, whereas PPARgamma 108 amino acid sequence showed much less similarity with non-fish PPARgamma. According to both semi-quantitative RT-PCR and real-time RT-PCR, PPARalpha mRNA predominates in white muscle, heart and liver and PPARbeta is more expressed in testis, heart, liver, white muscle and trunk kidney. PPARgamma was only detected in trunk kidney and liver by real-time RT-PCR and also in spleen by semi-quantitative RT-PCR. PPARbeta seems to be the most strongly expressed isotype, whereas PPARgamma shows a much weaker global expression.     

PPAR expression and function during vertebrate development

The peroxisome proliferator activated receptors (PPARs) are ligand activated receptors which belong to the nuclear hormone receptor family. As with other members of this superfamily, it is thought that the ability of PPAR to bind to a ligand was acquired during metazoan evolution. Three different PPAR isotypes (PPARalpha, PPARbeta, also called 6, and PPARgamma) have been identified in various species. Upon binding to an activator, these receptors stimulate the expression of target genes implicated in important metabolic pathways. The present article is a review of PPAR expression and involvement in some aspects of Xenopus laevis and rodent embryonic development. PPARalpha and beta are ubiquitously expressed in Xenopus early embryos but become more tissue restricted later in development. In rodents, PPARalpha, PPARbeta and PPARgamma show specific time- and tissue-dependent patterns of expression during fetal development and in the adult animals. PPARs are implicated in several aspects of tissue differentiation and rodent development, such as differentiation of the adipose tissue, brain, placenta and skin. Particular attention is given to studies undertaken by us and others on the implication of PPARalpha and beta in rodent epidermal differentiation.     

Cloning, sequencing, and characterization of CYP1A1 cDNA from leaping mullet (Liza Saliens) liver and implications for the potential functions of its conserved amino acids

A 2,037 bp CYP1A1 cDNA (GenBank AF072899) was cloned through screening of a lambdaZipLox cDNA library constructed from the liver of a leaping mullet (Liza saliens) fish captured from Izmir Bay on the Aegean coast of Turkey using rainbow trout CYP1A1 cDNA as a probe. This clone has a 130 bp 5'-flanking region, a 1,563 bp open reading frame (ORF) encoding a 521-amino acid protein (58,972 Da), and a 344 bp 3'-untranslated region without a poly (A) tail. Alignment of the deduced amino acids of CYP1A1 cDNAs showed 58% and 69-96% identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1 cDNA was from a single-copy gene. Based on the comparison with CYP1A1 genes reported for fish and mammals, the leaping mullet CYP1A1 gene is probably split into 7 exons. The intron insertion sites were predicted. Alignment of the CYP1A1 cDNA encoded amino acids from 13 fish and 7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates. The possible associated secondary structure; conserved motifs and substrate-binding sites were discussed. The phylogenetic relationships of CYP1A1s among 13 fish species were analyzed by a distance method.     

Peroxisome proliferator-activated receptors and skin development

PPARs are nuclear hormone receptors. PPAR subtypes (alpha, gamma, delta, the latter a xPPARbeta homologue) were initially investigated in skin because of their known role in regulating lipid metabolism. Studies adding specific PPAR ligand activators to cultured skin or skin cells are compatible with the concepts that PPARalpha activation mediates early lipogenic steps common to the function of both skin epidermal cells (keratinocytes) and sebaceous cells (sebocytes), PPARgamma activation plays a unique role in stimulating sebocyte lipogenesis, and PPARdelta activation may contribute to lipid biosynthesis in both sebocytes and keratinocytes under certain circumstances. Epidermal keratinocytes appear to express small amounts of PPARalpha and PPARdelta mRNA and a trace of PPARgamma mRNA which is up-regulated with differentiation. Sebocytes express all subtypes; PPARgamma gene expression excedes that in epidermis. The emerging data on PPAR protein expression suggests that epidermis normally expresses predominantly PPARalpha, while sebocytes express more PPARgamma than PPARalpha. These expression patterns may change during hyperplasia, differentiation and inflammation. Gene disruption studies in mice are compatible with a contribution of PPARalpha to skin barrier function, suggest that PPARgamma is necessary for sebocyte differentiation, and indicate that PPARdelta can ameliorate inflammatory responses in skin. PPARs appear to play a role in keratinocyte synthesis of the lipids that they export to the intercellular space to form the skin permeability barrier. They also appear to be important for sebocyte formation of the intracellular fused lipid droplets that constitute the holocrine secretion of the sebaceous gland. In addition, they may play roles in keratinocyte growth and differentiation and the inhibition of skin inflammation by diverse mechanisms not necessarily related to fat metabolism.     

SAR-oriented discovery of peroxisome proliferator-activated receptor pan agonist with a 4-adamantylphenyl group as a hydrophobic tail

3-(4-Alkoxyphenyl)propanoic acid derivatives were prepared as candidate peroxisome proliferator-activated receptor (PPAR) alpha/delta/gamma pan agonists, based on our previous SAR studies directed toward the development of subtype-selective PPAR agonists. Those studies indicated that the steric bulkiness of substituents introduced at the distal benzene ring had an important influence on PPAR activity. The finding that a 4-adamantyl derivative exhibited not only PPARalpha/delta activity but also significant PPARgamma activity prompted us to search for structurally novel phenylpropanoic acid derivatives with more potent adipocyte differentiation activity than the well-known PPARgamma agonist, rosiglitazone, as well as well-balanced PPARalpha and PPARdelta agonistic activities. A representative phenylpropanoic acid derivative (12) bearing a 4-adamantylphenyl substituent proved to be a well-balanced PPAR-pan agonist with activities to regulate the expression of genes involved in lipid and glucose homeostasis, and should be useful as a candidate drug for the treatment of altered PPAR function.     

Expression of peroxisome proliferator-activated receptors in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

Peroxisomes increase in size and number in responsive animals ranging from mammals to marine mussels and fish species when treated with certain compounds named peroxisome proliferators. This phenomenon, known as peroxisome proliferation, is mediated by nuclear receptors termed peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described (alpha, beta, and gamma) and in mammals PPARalpha is mainly expressed in tissues that catabolize fatty acids, PPARbeta is ubiquitously distributed, and PPARgamma is mainly expressed in the adipose tissue and immune system. The aim of this study was to analyze the tissue distribution of different PPAR subtypes in zebrafish Danio rerio using commercially available antibodies against PPARalpha, PPARbeta, and PPARgamma. In western blots, specific bands were detected at about 58 kDa for PPARalpha and PPARbeta. For PPARgamma the band was detected at 56 kDa. Similar results were obtained in mouse liver homogenates used as positive control, indicating the specificity of the antibodies. Immunohistochemistry was performed in paraformaldehyde-fixed tissue using either microwave or microwave plus trypsin pretreatment for antigen retrieval. In zebrafish, PPARalpha was expressed mainly in liver parenchymal cells, proximal tubules of kidney, enterocytes, and pancreas. PPARbeta showed a widespread distribution and was expressed in the liver, proximal and distal tubules and glomeruli of the kidney, pancreas, enterocytes and smooth muscle of the intestine, skin epithelium, lymphocytes, and male and female gonads. PPARgamma expression was weak in pancreatic cells, intestine, and gonads for both pretreatments. Most of the signal detected was cytoplasmic; only in the cases of PPARalpha and PPARbeta was some nuclear labeling detected in the liver. In mouse tissues, the distribution of PPAR subtypes was similar to that described previously for rats. Our results demonstrate that all three distinct PPAR subtypes are present in zebrafish. The tissue and cellular distribution of PPAR subtypes in zebrafish resembled partly that described before in mammals. Further studies are needed to decipher the functions of PPAR subtypes in zebrafish and other aquatic organisms and particularly their role in regulation of metabolic responses to xenobiotic exposure.     

Differential responses of PPARalpha, PPARdelta, and PPARgamma reporter cell lines to selective PPAR synthetic ligands

To characterize the specificity of synthetic compounds for peroxisome proliferator-activated receptors (PPARs), three stable cell lines expressing the ligand binding domain (LBD) of human PPARalpha, PPARdelta, or PPARgamma fused to the yeast GAL4 DNA binding domain (DBD) were developed. These reporter cell lines were generated by a two-step transfection procedure. First, a stable cell line, HG5LN, expressing the reporter gene was developed. These cells were then transfected with the different receptor genes. With the help of the three PPAR reporter cell lines, we assessed the selectivity and activity of PPAR agonists GW7647, WY-14-643, L-165041, GW501516, BRL49653, ciglitazone, and pioglitazone. GW7647, L-165041, and BRL49653 were the most potent and selective agonists for hPPARalpha, hPPARdelta, and hPPARgamma, respectively. Two PPAR antagonists, GW9662 and BADGE, were also tested. GW9662 was a selective PPARgamma antagonist, whereas BADGE was a low-affinity PPAR ligand. Furthermore, GW9662 was a full antagonist on PPARgamma and PPARdelta, whereas it showed partial agonism on PPARalpha. We conclude that our stable models allow specific and sensitive measurement of PPAR ligand activities and are a high-throughput, cell-based screening tool for identifying and characterizing PPAR ligands.