Comparative analysis of early embryonic sunflower cDNA libraries

To gain information concerning cell functions and activities during sunflower embryogenesis, an expressed sequence tag (EST) approach was used to analyse gene expression in the early stages of sunflower embryos development. Confocal microscopy observations of whole-mounted embryos allowed us to identify precisely the major steps of the zygotic embryonic development. A time-course analysis was then employed to collect the embryonic material. Three cDNA libraries were constructed from microdissected embryos, and three other cDNA libraries were created using a classical day after pollination schedule. A total of 7106 ESTs were produced and assembled. The total number of putative different genes represents about 43.1 (3064 tentative contigs and singlets) of the analysed sequences. The unigenes that showed similarity to proteins with known or predicted functions (50.3) were classified into 15 different functional categories. The functional profiles were found to be quite similar for all studied embryo stages but statistical analysis revealed that successive and coordinate sets of genes are expressed at each embryonic stage. The analysis allowed us to identify abundant and differentially expressed genes at the early stages of embryos development as well as some putatively interesting genes, showing strong similarities with genes playing key roles in plant and animal embryogenesis. The data presented in this study not only provide a first global overview of the genes expression profile during sunflower embryogenesis but also represent an original and valuable tool for developmental genomics studies on exalbuminous dicots.     

三角帆蚌GPX基因cDNA全序列的克隆及其编码蛋白的结构分析

根据本实验室构建的三角帆蚌cDNA文库中已标注的EST序列, 利用cDNA末端快速扩增法(RACE)克隆了三角帆蚌(Hyriopsis cumingii)谷胱甘肽过氧化物酶(Glutathione peroxidase, GPX)基因cDNA全序列。序列分析表明, 该基因cDNA序列全长1286 bp, 包括5′端非翻译区(Untranslated Region) 39 bp、3′端非翻译区659 bp和开放阅读框(Open reading frame, ORF)588bp, 共编码195个氨基酸, 分子量约为22.2 kDa, 理论等电点为8.44, 属于含硒类GPX。该氨基酸序列具有GPX所有亚型中均高度保守的3个环状结构, 对酶的三级结构起稳定作用。在线分析结果表明: GPX的氨基酸序列不存在明显的疏水区, 也不存在信号肽序列。氨基酸相似性对比结果显示, 三角帆蚌GPX氨基酸序列与脊椎动物GPX-2及GPX-1的序列相似度较高, 为73.1%-80.8%, 与其他型GPX相似度较小, 相似度低于60%。构建的系统进化树显示三角帆蚌GPX与其他几种鱼类GPX聚为一类, 与其他已发表的几种软体动物GPX相距较远, 推测本实验克隆的三角帆蚌GPX基因和已发表的软体动物不属于同一种GPX类型。     

目的基因全长cDNA合成及克隆技术进展

在基因克隆、基因功能和表达调控研究中 ,获得目的基因尤其是表达丰度低的基因全长cDNA仍然是一个技术难题。本文对近年来有关反转录过程中影响全长cDNA合成的因素与对策的研究进行了总结 ,特别对消除mRNA二级结构阻碍的方法及其效果、较高反应温度和金属离子对模板mRNA完整性的影响、RNA分子伴侣在反应温度不变的情况下改善cDNA第一链质量的效果等作了较为详细的介绍。此外 ,还介绍了确保具有完整5′端的cDNA进入后续研究的几种新方法。     

cDNA cloning and functional characterization of a novel splice variant of c-Cbl-associated protein from mouse skeletal muscle

c-Cbl-associated protein (CAP) is an SH3-containing adapter protein that binds to the proto-oncogene c-Cbl. Recent work suggests that signaling through these molecules is involved in the regulation of insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Skeletal muscle is the major site of insulin-stimulated glucose disposal but there have been no reports of CAP function in this tissue. Using RT-PCR of mouse skeletal muscle RNA, we discovered a novel splice variant of CAP (CAPSM; GenBank Accession No. AF521593) that is different from the adipocyte form by inclusion of a novel 168 bp fragment. This fragment encodes a peptide sequence that shows very high similarity with exon 25 of the human homologue of CAP (SORBS1). To understand the function of CAPSM in glucose uptake regulation, L6 myotubes were transfected with either CAPSM or a truncated CAPSM devoid of all three SH3-binding domains (CAPDeltaSH3), which prevents CAP association with c-Cbl. Transfection with CAPDeltaSH3 decreased insulin-stimulated 2-deoxyglucose (2-DG) uptake and reduced c-Cbl phosphorylation. In contrast, transfection of L6 myotubes with CAPDeltaSH3 had no effect on dinitrophenol (DNP)- or hypoxia-stimulated glucose uptake, stimuli that work through insulin-independent mechanisms for the regulation of glucose uptake. These data demonstrate the existence of a novel CAP isoform expressed in skeletal muscle, and suggest the involvement of the CAP/Cbl pathway in the regulation of insulin-stimulated glucose uptake in L6 myotubes.     

百合ACC氧化酶基因全长cDNA的克隆及序列分析

以亚洲百合Polyanna(Liliumspp.)花瓣为材料,根据已报道的百合ACC氧化酶基因片段设计1对末端扩增特异引物,采用RACE方法,获得百合ACC氧化酶基因的全长cDNA(GenBank登录号为EU296623).该cDNA全长1 152 bp,具有一个954 bp的开放阅读框,编码318个氨基酸.Blast搜索结果显示,百合ACC氧化酶基因核苷酸序列与其它植物已报道的ACC氧化酶基因具有71%~82%的相似性,氨基酸序列有70%~87%的相似性,聚类分析表明,与单子叶植物百合科郁金香首先聚类,其次与双子叶植物聚类,最后与单子叶禾本科和兰科植物聚类.     

Compositional properties of human cDNA libraries: practical implications

The strikingly wide and bimodal gene distribution exhibited by the human genome has prompted us to study the correlations between EST-counts (expression levels) and base composition of genes, especially since existing data are contradictory. Here we investigate how cDNA library preparation affects the GC distributions of ESTs and/or genes found in the library, and address consequences for expression studies. We observe that strongly anomalous GC distributions often indicate experimental biases or deficits during their preparation. We propose the use of compositional distributions of raw ESTs from a cDNA library, and/or of the genes they represent, as a simple and effective tool for quality control.     

苎麻纤维素合酶基因BnCesA1全长cDNA的克隆与表达分析

苎麻是中国的传统纤维作物,能够生产最长的自然纤维。本研究旨在克隆苎麻纤维素合酶基因BnCesA1全长编码序列,对其表达模式进行分析。以已知的苎麻纤维素合酶基因序列(DQ077190)为基础,设计5’RACE引物,以湘苎三号为材料,得到了BnCesA1的5’端,拼接后得到了BnCesA1的全长序列,并从湘苎三号的cDNA中成功克隆到包括BnCesA1全部编码序列的cDNA序列。扩增得到的BnCesA1基因cDNA为3253bp,编码区3246bp,编码含1082个氨基酸的多肽。通过对这个基因进行核酸序列和蛋白结构域分析表明,BnCesA1和毛果杨、欧美山杨、巨桉、大叶相思等其他物种的纤维素合酶基因都有很高的同源性,根据得到的BnCesA1的5’端设计特异性表达检测引物,分析其在湘苎三号苎麻品种各组织中的表达情况,结果显示BnCesA1在所检测的各组织中均有表达,表达量为茎皮>叶>顶芽>根。本研究首次克隆到苎麻中编码全长蛋白的纤维素合酶基因,并且苎麻BnCesA1在茎皮中高表达提示该基因可能在苎麻韧皮纤维合成中有重要作用。     

Long C terminal splice variant CaV2.2 identified in presynaptic membrane by mass spectrometric analysis

Ca_V2.2 voltage-gated calcium ­channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with Ca_V2.2 in purified presynaptic terminal membranes. A number of known and new Ca_V2.2-associated proteins were identified, but not the channel itself. Here we set out to explore this anomaly. As previously, we used antibody Ab571 to capture the channel from purified synaptosome membrane lysate. We prepared a brain membrane lysate enriched for presynaptic active zones using standard methods to fractionate purified synaptosomes. These were osmotically lysed to generate a fraction enriched in presynaptic surface membranes. The lysate was solubilized in modified RIPA buffer and was passed over anti-Ca_V2.2 antibody covalently bonded to immunoprecipitation beads. Captured complexes on the beads were then stripped of weakly-bound proteins by exposure to high salt to enrich the channel fraction. Proteins remaining bound to the sample were recovered in high concentration urea and the sample was subjected to standard enzyme digestion and MS analysis. We identified 12 distinct Ca_V2.2 peptides, but no other ion channel peptides, in the lysate-exposed bead sample but no other ion channel peptides were recovered. Interestingly one of the channel peptides was derived from the alternatively spliced, long-C terminal region. Hence, confidence in identification of Ca_V2.2 was beyond reasonable doubt. The identification of the long-splice Ca_V2.2 provides compelling evidence that this variant is targeted to the presynaptic terminal, as we and others have suggested.     

致蛋鸡血管瘤J亚群禽白血病病毒cDNA全序列分析

【目的】了解近年来我国商品蛋鸡群中以血管瘤为主要表型的J亚群禽白血病病毒(Avian Leukosis Virus subgroup J,ALV-J)的分子生物学特性,为控制ALV-J在鸡群中流行提供基础资料。【方法】采用PCR扩增和序列分析技术,对分离自血管瘤或者血管瘤与髓样细胞瘤(Myeloid Leukosis,ML)并存的3株蛋鸡ALV-J毒株前病毒DNA的全序列及3株商品蛋鸡血管瘤型分离毒和1株商品蛋鸡ML型分离毒的致瘤关键性序列进行研究。【结果】来自血管瘤或者血管瘤与ML并存的商品蛋鸡分离毒株与来自肉鸡分离毒株的全序列差异明显,在遗传进化树上分属两个大的分支;研究发现商品蛋鸡血管瘤及ML混合病例分离毒JS09GY3与JS09GY6株的引物结合位点(Primer Binding Site,PBS)-Leader中出现极为罕见的连续19bp的插入突变,其与劳斯相关病毒1(Rous Associated Virustype1,RAV-1)、劳斯相关病毒2(Rous Associated Virustype2,RAV-2)及劳斯肉瘤病毒施密特-鲁宾二氏[Rous sarcoma virus(strain Schmidt-RuppinB),RSV-SRB]毒株序列相同;通过对U3区调控元件的分析,发现血管瘤商品蛋鸡病例分离毒NHH与JS09GY5的U3区各发生1处连续序列缺失,出现了极为独特的c-Est-1、TCF11及C/EBP结合位点,这些调控元件可能与病毒的致肿瘤特性相关;所测的5株血管瘤商品蛋鸡分离毒均保留完整E元件,而所有肉鸡分离毒的E元件均发生了几乎相同的大部分序列缺失;首次发现血管瘤商品蛋鸡分离毒JS09GY3的E元件中有11bp的连续插入序列。【结论】商品蛋鸡血管瘤型ALV-J与肉鸡分离毒在全序列上差异明显,U3、DR1和E元件等区域有一部分特殊的突变与毒株的宿主类型和肿瘤表型有一定关系,其功能尚需进一步研究。而血管瘤型、髓细胞瘤型ALV-J可能是ALV-J与其它反转录病毒的重组毒。     

Molecular analysis of a new splice variant of the human melanocortin-1 receptor.

The primary hormonal regulator of pigmentation is melanocyte stimulating hormone derived from proopiomelanocortin by proteolytic processing. The melanocortin-1 receptor serves a key role in the regulation of pigmentation. We describe the identification of the first intron within a melanocortin receptor. A new melanocortin-1 receptor isoform, generated by alternative mRNA splicing, encodes an additional 65 amino acids at the predicted intracellular, C-terminal tail of the melanocortin-1 receptor. When expressed in heterologous cells, the new spliced form of the melanocortin-1 receptor (melanocortin-1 receptor B) appears pharmacologically similar to the non-spliced melanocortin-1 receptor. Melanocortin-1 receptor B is expressed in testis, fetal heart and melanomas.     

cDNA cloning and expression of the mouse Na/H antiporter (NHE-1) and a potential splice variant

A cDNA for the Mus musculus Na/H exchanger-isoform 1 (NHE-1) was identified in a BALB/c myoblast library by its hybridization to rat NHE-1 sequences. Analysis of the clone showed it to display extensive homology with NHE-1 clones from other mammalian species; however, the region of interspecific homology was abruptly interrupted in the midst of the open reading frame by 166 bp of unrelated sequence. This extra sequence is likely to be an unspliced intron 9. Aside from the retained intron 9, the NHE-1 cDNA clone is otherwise fully processed, with all of the other ten introns removed and containing a poly(A) tract. From PCR results this variant represents a significant but minor population of NHE-1 RNAs. The variant message does associate with polysomes thereby suggesting it to be translated into protein. The location of the retained intron in the carboxy terminus of the protein is such that its translation would produce a protein predicted to be still capable of effecting Na and H translocation but whose regulatory features would be markedly altered.Amino acid sequence comparison of the mouse NHE-1 (derived from the fully processed message) with that of other mammalian species demonstrated two exceptionally divergent regions; the C-terminal cytoplasmic tail (residues 750-790), containing a region of 6-8 contiguous acidic amino acids variably composed of aspartate and glutamate residues, and the N-terminal extracellular domain that includes an N-linked glycosylation site (residues 60-80).     

生物信息学在新基因全长cDNA电子克隆中的应用

新基因全长cDNA序列的获得常常是生物学工作者面临的难题,电子克隆是利用生物信息学手段得到新基因全长cDNA序列的新方法。介绍了电子克隆的方法及其生物信息学在其间的具体应用,并概述了一些生物信息学在序列分析中的应用。     

Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection

Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.     

Bovine hexokinase type I: Full-length cDNA sequence and characterisation of the recombinant enzyme

This study reports the revised and full-length cDNA sequence of bovine hexokinase type I obtained from bovine brain. Since dissimilarities have been observed between the published bovine hexokinase type I coding sequence (GenBank accession no. M65140) (Genomics 11: 1014-1024, 1991) and an analysed portion of bovine hexokinase type I gene, the entire open reading frame was re-sequenced and the ends of cDNA isolated by rapid amplification of cDNA ends. The coding sequences, when compared with the published bovine hexokinase type I, contained a large number of mismatches that lead to changes in the resulting amino acid sequence. The revisions result in a hexokinase type I cDNA of 3619 bp that encodes a protein of 917 amino acids highly homologous to human hexokinase type I. The expression of the recombinant full-length enzyme demonstrated that it was a catalytically active hexokinase. When characterised for its kinetic and regulatory properties, it displayed the same affinity for glucose and MgATP as the human hexokinase type I and was inhibited by glucose 6-phosphate competitively versus MgATP. The production of the N- and C-terminal recombinant halves of the enzyme followed by comparison with the full-length hexokinase indicated that the catalytic activity is located in the C-terminal domain. (Mol Cell Biochem 268: 9–18, 2005)