Evolutionary Comparison of Two Combinatorial Regulators of SBP-Box Genes,MiR156 and MiR529, in Plants

A complete picture of the evolution of miRNA combinatorial regulation requires the synthesis of information on all miRNAs and their targets. MiR156 and miR529 are two combinatorial regulators of squamosa promoter binding protein-like (SBP-box) genes. Previous studies have clarified the evolutionary dynamics of their targets; however, there have been no reports on the evolutionary patterns of two miRNA regulators themselves to date. In this study, we investigated the evolutionary differences between these two miRNA families in extant land plants. Our work found that miR529 precursor, especially of its mature miRNA sequence, has a higher evolutionary rate. Such accelerating evolution of miR529 has significantly effects on its structural stability, and sequence conservation against existence of itself. By contrast, miR156 evolves more rapidly in loop region of the stable secondary structure, which may contribute to its functional diversity. Moreover, miR156 and miR529 genes have distinct rates of loss after identical duplication events. MiR529 genes have a higher average loss rate and asymmetric loss rate in duplicated gene pairs, indicating preferred miR529 gene losses become another predominant mode of inactivation, that are implicated in the contraction of this family. On the contrary, duplicated miR156 genes have a low loss rate, and could serve as another new source for functional diversity. Taken together, these results provide better insight into understanding the evolutionary divergence of miR156 and miR529 family in miRNA combinational regulation network.     

MouseSprr2Genes: A Clustered Family of Genes Showing Differential Expression in Epithelial Tissues

Small proline-rich (SPR) proteins are structural components of the cornified cell envelope of stratified squamous epithelia. They are subdivided into three families, i.e., SPR1, SPR2, and SPR3, of which the SPR2 family is the most complex. To understand the significance of this complexity, we have isolated 11 mouseSprr2genes, constructed a provisional physical map of theSprr2locus on mouse Chromosome 3, and examined the expression patterns of theSprr2genes in mouse epithelial tissues. The 11Sprr2sequences are highly conserved with a central domain containing a variable number of repeats.In situhybridization showed theSprr2expression to be confined to epithelia. RT-PCR using primers specific for each of the 11Sprr2members demonstrated varying degrees of expression among the individualSprr2members in different tissues. The correlation between the physical location of the genes in theSprr2locus and their expression patterns suggests multiple levels of controlled expression.     

脊椎动物DMRT基因家族的系统发生及同线性分析

利用本室克隆的DMRT／Dmrt基因以及多个基因数据库中收集的DMRT／Dmrt基因全(或部分)序列,构建了DMRT／Dmrt基因的系统发生树,DMRT／Dmrt基因明显地聚为7类(DMRT／Dmrt1-DMRT／Dmrt7)。以已克隆的斑马鱼Dmrt1基因序列及数据库中人和小鼠的DMRT／Dmrt基因序列,结合基因组数据的比对分析,发现脊椎动物DMRT／Dmrt基因家族成员在基因组中的定位呈现高度保守的同线性,主要集中于两大同线群,即DM-RT／Dmrt1-3和DMRT／Dmrt5∽6。     

A Comprehensive Analysis of the Phylogeny,Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis,an Endangered Reptile Species

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 C_λ genes, each preceded by a J_λ gene, and 86 potentially functional V_λ genes upstream of (J_λ-C_λ)_n. The Igκ locus contains a single C_κ gene, 6 J_κs and 62 functional V_κs. All V_L genes are classified into a total of 31 families: 19 V_λ families and 12 V_κ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed V_λ and V_κ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.     

水稻组蛋白H_3基因的分离和组织结构分析

用噬菌斑原位杂交法,从一个水稻新品系的EMBL,基因文库中筛选到了两个组蛋白H,基因克隆,并制作了它们的限制性内切酶图谱,分析了它们的组织结构。其中λRH_3-1克隆包含2个组蛋白H_3基因区(分别为0.5kb和0.8kb的BamHI-EcoRI片段,两基因区距离5.8kb)。而λRH_3-2克隆只带有1个结构特异的H_3基因区(5.2kb的EcoRI-HindⅢ片段)。本文还就水稻组蛋白H_3基因在整个基因组中的组织结构特点进行了讨论。     

Genomic and Phylogenetic Analysis of the Human CD1 and HLA Class I Multicopy Genes

The human CD1 proteins belong to a lipid-glycolipid antigen-presenting gene family and are related in structure and function to the MHC class I molecules. Previous mapping and DNA hybridization studies have shown that five linked genes located within a cluster on human chromosome 1q22-23 encode the CD1 protein family. We have analyzed the complete genomic sequence of the human CD1 gene cluster and found that the five active genes are distributed over 175,600 nucleotides and separated by four expanded intervening genomic regions (IGRs) ranging in length between 20 and 68 kb. The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Some L1 sequences have acted as receptors for other subtypes or families of retroelements. Alu molecular clocks that have evolved during primate history are found distributed within the HLA class I duplicated segments (duplicons) but not within the duplicons of CD1. Phylogeny of the alpha3 domain of the class I-like superfamily of proteins shows that the CD1 cluster is well separated from HLA class I by a number of superfamily members including MIC (PERB11), HFE, Zn-alpha2-GP, FcRn, and MR1. Phylogenetically, the human CD1 sequences are interspersed by CD1 sequences from other mammalian species, whereas the human HLA class I sequences cluster together and are separated from the other mammalian sequences. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans. In contrast to the HLA class I genomic structure, the human CD1 duplicons are smaller in size, they lack Alu clocks, and they are interrupted by IGRs at least 4 to 14 times longer than the CD1 genes themselves. The IGRs seem to have been created as "buffer zones" to protect the CD1 genes from disruption by transposable elements.     

葡萄孤儿基因的表达分析和功能推测

孤儿基因为某物种基因组内特有的,在其它物种内没有同源基因存在的特殊基因.对物种孤儿基因的鉴定和分析已成为比较基因组学研究的一个新兴领域.本研究通过生物信息学的分析,着重研究了49个葡萄孤儿基因在各发育阶段和各器官中的特异表达特性、亚细胞定位预测和共表达基因群的生物学过程基因本体论(GO)分析.结果显示,35个基因在NCBI的EST数据库中存在EST表达证据,其中有7个基因具有器官表达特异性,且大部分在生殖器官中特异表达.通过亚细胞定位预测到大部分葡萄孤儿基因为分泌蛋白.GO分析表明葡萄孤儿基因的共表达基因群主要参与寡肽转运和跨膜运输等生物学过程,这与葡萄孤儿基因的亚细胞定位预测结果相一致.本文研究结果将为葡萄孤儿基因的进一步功能分析提供重要研究基础.     

Genomic Organization of the Mammalian SLC14a2 Urea Transporter Genes

Urea transporters encoded by the UT-A gene play fundamental roles in the kidney and possibly other tissues. Knowledge of the genomic organization of the mouse, rat and human UT-A genes has enabled the engineering of transgenic and knockout animals and these have helped refine our understanding of the role of UT-A proteins. This review summarizes the published work that has accrued on the structure and regulation of these genes. It also documents a novel cDNA, human UT-A3, which has enabled a major refinement of the human UT-A gene structure. This and other information contained in this review should prove useful for future comparative genomic analysis, studies addressing gene regulation and for the engineering of transgenic and knockout animal strains.     

小麦光温敏核雄性不育相关基因的G-box家族引物差式分析

用G-box家族引物对小麦光温敏核雄性不育系农大3338在可育与不育光温条件下进行mRNA差异显示,结果表明,在育性转换时期,这两种条件下的基因表达存在显著差异。回收了12个质的差异片段并进行反Northern印迹杂交验证,然后对5个阳性克隆片段HT1-G10、HT1-G3、HT2-G2、HT1-G4和HT2-G5进行了测序,同源比较显示：HT1-G10与小麦（Triticum aestivum）叶绿体基因rbcL和atpB的部分序列高度同源（96％）;HT1-G3与小麦（Triticum aestivum）组蛋白H2A基因高度同源（88％）;另3个片段为新基因片段。对这些基因片段的分析为揭示光温敏核雄性不育的发育机理提供了一些有效证据。     

Vicilin Genes of Vigna luteola: Structure, Organization, Expression, and Variation

Two different but related sequences that encode Vigna luteola 7S vicilins were isolated and characterized. The sequences differ by two nucleotide substitutions, each of which results in an amino acid replacement. This low level of divergence suggests that a recent gene duplication has occurred. Both variants are expressed in cDNA populations; therefore, neither gene is a pseudogene. Both copies were present in all individuals (72) analyzed using real-time PCR and TaqMan probes. Segregation was not observed. The two sequences are not independent alleles. Vicilin genomic sequences of 11 specimens from six geographic locations were determined. No polymorphic sites were identified in either of the two gene copies. This lack of polymorphism suggests that either a population bottleneck or selection has occurred. The genetic structure, expression patterns, and protein composition of the V. luteola vicilins were compared to those of other legume vicilins.     

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization,Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.     

葡萄NIP基因家族的鉴定与表达分析

类根瘤菌26膜内在蛋白(nodulin 26 like intrinsic proteins,NIPs)是水通道蛋白的亚类,在植物营养获取和胁迫应答过程中发挥着重要作用。该研究利用多种生物信息学软件,对葡萄NIP家族基因进行分析,并采用RT PCR方法克隆得到4个NIP家族基因,利用qRT PCR方法分析非生物胁迫下NIP基因的表达特征。结果显示：(1)在葡萄基因组中,共鉴定到8个NIP基因,分布于葡萄4条染色体上,主要定位在质膜中;结构上含有6个跨膜结构域和两个典型的保守结构域NPA;氨基酸序列中存在很多个可能的磷酸化位点。(2)进化分析表明葡萄和拟南芥NIP基因具有较高的同源性,基因结构包含外显子数4~6个,保守基序种类和数量相似;基因启动子上游2 kb包含多种应答逆境和激素的顺式调控元件,其数量差异可能与基因本身功能相关。(3)NIP家族基因在不同组织中表达水平差异较大,多数成员在叶中表达水平较高,在茎中较低;成功克隆得到4个葡萄VvNIP基因,其长度分别为789 bp、606 bp、897 bp、789 bp,分别编码262、201、298、293个氨基酸。(4)qRT PCR结果显示,不同胁迫处理下NIP基因在葡萄叶片中的表达水平不同：低温处理下葡萄NIP基因大多呈显著下调表达;盐胁迫下,除VvNIP2 1、VvNIP4 2外其余家族基因均呈下调表达;干旱胁迫下VvNIP4 2显著上调。研究表明,VvNIP基因对多种胁迫均有响应,为葡萄逆境胁迫机制研究提供了参考。     

Molecular Cloning, Genomic Organization, and Expression of a Testicular Isoform of Hormone-Sensitive Lipase

By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSL_tes. Due to an addition of amino acids at the NH₂-termini, rat and human HSL_tesconsist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSL_adi). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSL_adi. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSL_adisequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M_r≈ 120,000) that exhibited catalytic activity similar to that of HSL_adi. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells.     

Chromosomal Organization,Evolutionary Relationship,and Expression of Zebrafish GnRH Family Members

Summary Multiple forms of gonadotropin-releasing hormone (GnRH) are found in different vertebrates. In this study, we have cloned cDNA encoding the full-length gnrh3 and gnrh2 from zebrafish brain and characterized their structure and expression patterns. We performed phylogenetic analysis and compared conserved syntenies in the region surrounding the GnRH genes from human, chicken, pufferfish, and zebrafish genores. The gnrh3 and gnrh2 genes were mapped to LG17 and LG21, respectively. The zebrafish genome appears to lack an ortholog to human GNRH1, and the human genome appears to lack an ortholog of gnrh3. Expression of gnrh3 began in the olfactory pit at 24–26 h postfertilization and expanded to the olfactory bulb during early larval stage. Expression of gnrh2 is always in the midbrain. In addition, GnRH is also expressed in boundary cells surrounding seminiferous cysts of the testis. Thus, this detailed phylogenetic, chromosomal comparison, and expression study defines the identity and the evolutionary relationship of two zebrafish gnrh genes. We propose a model describing the evolution of gnrh genes involving ancestral duplication of the genes followed by selective loss of one gene in some teleosts.     

葡萄NCED基因家族进化及表达分析

9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物体内ABA生物合成的关键限速酶, 参与植物对干旱、外源ABA和高盐的响应过程, 降低环境胁迫对植株的危害。基于全基因组鉴定分析葡萄(Vitis vinifera) NCED基因家族成员, 探讨各成员的物种进化关系及各个基因成员在不同组织中的时空表达模式及对干旱、ABA和高盐(NaCl)胁迫的响应, 为进一步揭示该基因家族成员的生物学功能奠定基础。在葡萄基因组中共发现12个NCED基因。其推测的编码蛋白质长度在510 (VvNCED2)-625 aa (VvNCED10)之间。VvNCED蛋白的分子量最大值是70.53 kDa (VvNCED10), 最小值是57.85 kDa (VvNCED2)。在从祖先基因分化之后, 葡萄NCED基因发生了5次复制事件, 同时有2次丢失事件。NCED1/2、NCED3/4、NCED6/7和NCED9/10基因对被认为是通过片段复制产生。上述4对复制基因复制时间分布在3.08-120.0百万年前, 晚于单双子叶植物分化的时间。与对照相比, VvNCED1在ABA处理48小时后显著上调(72.1%), 而VvNCED2显著下调(84.0%)。VvNCED6只在干旱处理14、21和28天的根系中表达量高于对照, 分别为对照的2.49、1.05和1.09倍。VvNCED7只在干旱处理14天的根系中表达量高于对照, 为对照的1.07倍。在ABA处理72小时后, VvNCED3表达量较对照显著下调(59.5%), 而VvNCED4较对照显著上调(169.9%)。VvNCED3/VvNCED4分别在NaCl处理24和48小时出现显著性峰值, 较对照分别上调219.2%和114.4%。保守结构域不同组成和不同胁迫处理下差异表达模式是NCED蛋白发生功能分化的基础。推测NCED在进化过程中发生的功能分化有利于复制事件的发生。