Structure of the pro alpha 2 (I) collagen gene

Fifty-four kilobase pairs (kbp) of cloned chicken DNA containing the entire 38-kbp pro alpha 2 (I) collagen gene have been isolated and characterized. DNA sequence analysis of a select 4 kbp of the gene has precisely described 14 exons which comprise one-third of the sequences encoding the triple-helical domain of the collagen protein. These exons range in size from 45 to 108 base pairs (bp), are all multiples of the 9 bp that code for the repeating triplet, Gly-X-Y, and have an average size of 70 bp. About 50 introns interrupt this gene. Nevertheless, introns do not separate the coding sequences for the ends of the central triple-helical structural domain and the ends of the propeptide domains.     

Conservation of the sizes for one but not another class of exons in two chick collagen genes

Type III collagen is often found in the same tissues as type I collagen, yet the function and nature of the fibrils formed by the two collagens differ markedly. To understand the evolutionary history of the collagen gene family in more detail, we isolated the gene for type III collagen and compared its structure with that of the gene for alpha 2(I) collagen. This comparison points to a remarkable conservation in the size distribution of the exons coding for the helical part of these two collagen polypeptides: equivalent amino acid segments in the helical domain of each polypeptide are encoded by exons of equal sizes in each gene. This suggests that after the interstitial collagen genes had been duplicated from a common ancestor about 2-5 X 10(8) years ago, no recombinations between these exons were tolerated, although the same recombinational phenomena must have played an important part in shaping the structure of the progenitor for these genes. This fixation of the size distribution of the exons which code for the interstitial collagen helical domains is found despite the persistence in these exons of sequence elements that should have favoured recombinational rearrangements, and contrasts with the variations in the pattern of sizes of some exons coding for the amino and carboxyl propeptides of these collagens.     

Human pro alpha 1(I) collagen gene structure reveals evolutionary conservation of a pattern of introns and exons

The collagens represent an interesting example of a structurally related but genetically distinct family of proteins. Type I, the most abundant of the vertebrate collagens, comprises two pro alpha 1(I) chains and one pro alpha 2(I) chain, each containing terminal propeptides and a central domain of 338 (Gly, X, Y) repeats. The structure of the chicken pro alpha 2(I) gene shows an intriguing relationship between exon organization and the arrangement of (Gly, X, Y) repeats (see ref. 2 for review). This has led to the suggestion that the collagens evolved from a common ancestral unit of 54 base pairs (bp). Here we present the structure of the entire human pro alpha 1(I) gene and compare this with the chicken pro alpha 2(I). The exon arrangement of the two genes is remarkably similar, although the human pro alpha 1(I) is more compact because of the shorter length of its introns. The data strongly support the notion that the type I genes have evolved from an ancestral multi-exon unit, and that once the gene was translated, a strong evolutionary pressure caused it to maintain this elaborate structure.     

Embryonic lethality caused by mutations in basement membrane collagen of C. elegans

Basement membranes are specialized forms of extracellular matrix with important functions in development. A major structural component of basement membranes is type IV collagen, a heterotrimer of two alpha 1(IV) and one alpha 2(IV) chains, which forms a complex, polygonal network associated with other basement membrane components. Here we report that the alpha 1(IV) collagen chain of Caenorhabditis elegans is encoded by the genetic locus emb-9. Mutations in emb-9 cause temperature-sensitive lethality during late embryogenesis. We have identified single nucleotide alterations that substitute glutamic acid for glycine in the triple-helical Gly-X-Y repeat region of the alpha 1(IV) collagen in three emb-9 mutant strains. These results are direct evidence that defects in basement membranes can disrupt embryonic development and form a basis for the genetic analysis of basement membrane function.     

Subtle structural alterations in the chains of type I procollagen produce osteogenesis imperfecta type II

Although the perinatal lethal form of osteogenesis imperfecta (OI type II) occasionally results from large rearrangements within the genes encoding type I collagen, most mutations are far more subtle. The complexity of the human collagen genes precludes cloning and sequencing each gene from every patient, and we have therefore developed an approach to localizing mutations at the protein level. We report here that cells cultured from 15 infants with OI type II synthesized both normal type I procollagen and a form that was unstable, poorly secreted and excessively modified. Abnormal procollagen from different strains was overmodified to different extents. The patterns of overmodification we observed are best explained by mutations that disrupt the Gly-X-Y sequence of pro alpha chains, and thus alter the rate of propagation of triple helix from COOH-terminus to NH2-terminus. As a consequence, a given mutation allows overmodification of all three chains in a molecule NH2-terminal to its position in the triple helix.     

小鼠胚胎脑特异表达的新基因bsg3的克隆及初步研究

通过消减差异筛选的方法克隆到一个在小鼠胚胎脑特异表达的基因bsg3(brain specific gene 3).它编码的蛋白与人的KIAA0961具有80.3%的同源性.bsg3基因包含一个1644bp的完整阅读框,编码一个含548个氨基酸,具有1个KRAB结构域(kruppel-associated box)和13个C2H2型锌指结构域的蛋白.该基因定位在小鼠第7号染色体上,包含5个外显子,4个内含子.以bsg3基因全长编码区为探针的原位杂交结果显示,bsg3在小鼠胚胎脑及鸡胚脑特异表达.半定量反转录聚合酶链式反应(RT-PCR)的结果表明,在成体小鼠的组织中,bsg3基因脑中表达也较强.这提示bsg3基因可能在小鼠脑发育中起着重要的作用.对bsg3基因时间和空间的表达模式的分析将有助于进一步揭示它在脑发育及功能维持中的作用.     

Cloning,sequencing of the HSC70 gene in Ctenopharyngodon idella

A cDNA encoding heat shock cognate protein 70(HSC70)was cloned from liver of grass carp(Ctenopharyngodon idella)(GenBank JF436930).This cDNA was found out to contain2 346 bp in length,including 1 950 bp of complete coding sequence encoding 649 amino acids(aa),plus 89 bp of 5′-UTR and 307 bp of3′-UTR.Analysis of its genomic structure revealed that its corresponding gene contained seven exons and six introns.Homology analysis indicated that it shared 99%of identity with HSC70 of breams and 86%of identity with HSP70 of Drosophila.Fluorescent RT-PCR analysis revealed that at 28℃,this gene was expressed in abdominal fat,muscle,intestines,brain,middle kidney,head kidney,gonads,swim bladder,liver,heart,spleen,gills,and fins with expression level in liver being the highest(p0.05),followed by that in the gonads;at 36℃,its mRNA expression level was increased at first but then decreased thereafter under heat shock stress,indicating that its expression can be regulated by heat shock.In conclusion,cloning and expression analysis identified a cDNA encoding a constitutive HSP70 gene that is expressed in many tissues of Ctenopharyngodon idella and its expression was down-regulated by heat shock.     

Polymorphism and absence of Leu-enkephalin sequences in proenkephalin genes in Xenopus laevis

The structures of the genes coding for the opioid peptide precursors proopiomelanocortin, proenkephalin (proenkephalin A) and prodynorphin (proenkephalin B), are known for some mammalian species. To gain insight into the evolutionary history of these precursors, we have examined the proenkephalin gene in the South African clawed toad, Xenopus laevis, which diverged from the principal line of vertebrate evolution some 350 Myr ago. The human proenkephalin gene consists of four exons, of which the main exon (exon IV) contains all known biologically active peptides--six Met-enkephalin sequences and one Leu-enkephalin sequence. We report here the primary structures of the putative main exons of two proenkephalin genes in X. laevis, each of which codes for seven Met-enkephalin sequences but no Leu-enkephalin, indicating that Met-enkephalin preceded Leu-enkephalin in the evolution of the proenkephalin gene. The organization of the main exons of the toad genes is remarkably similar to that of the human gene and conserved regions provide evidence for functionally significant structures. We also detect a polymorphism in one of the toad proenkephalin genes, mapping 1.5 kilobases (kb) 5' of the main exon; it is caused by an insertion/deletion of a 1-kb repetitive sequence which has the characteristics of a transposable element.     

短柄五加(Acanthopanax brachypus)rbcL基因的结构分析

克隆了含完整短柄五加rbcL基因的3.2kb EcoRI片段,测定了该基因的核苷酸序列.所测核苷酸序列总长度为1924bp,其中编码区1428bp,编码475个氨基酸的蛋白质.测定的基因5’上游区共278bp,包含原核性质-35区(TTGCGC),-10区(TACAAT)及类似真核的TATA box元件(TATATA).5’前导区长194bp,其中SD序列为GGAGG,紧邻起始密码子上游.测定的3’下游区共218bp,含2个相邻的转录后可形成茎环结构的反向重复序列.短柄五加rbcL基因编码区推导的氨基酸序列与烟草、菠菜、豌豆、苜蓿、玉米、水稻、松树、地钱、衣藻和Anacystis的同源性分别为93.5％、94.11％、94.53％、94.74％、89.68％、92.21％、92.21％、92.63％、87.58％和80.84％.本文还对不同植物rbcL基因的启动区及部分5’和3’非编码区进行了比较分析.     

小麦液泡质子焦磷酸酶基因EST的克隆与分析

通过RT-PCR的方法在矮苏3小麦的总cDNA中克隆到一个与大麦液泡质子焦磷酸酶基因(VP)高度同源的EST。以该序列为基础,利用生物信息学的方法构建了一个编码小麦VP蛋白的全长EST重叠群,长2764bp,其包含一个长2355bp的完整开放读码框(ORF),编码785个氨基酸多肽。通过Southern杂交,将该VP基因定位在小麦染色体的第7同源群上。     

尼罗罗非鱼Hepcidin基因结构与序列分析

Hepcidins是一类具有调节铁代谢功能的抗菌肽.利用RT-PCR、RACE和LA等技术,以尼罗罗非鱼[Oreochro-mis niloticus(Linnaens)]肝脏中分离和克隆到Hepcidin基因.其全长cDNA为505 bp(不包括polyA),5′端非翻译区有85 bp,3′非编码区为156 bp,阅读框为264 bp.其编码的氨基酸包括信号肽和前体肽等,前体肽进一步酶解产生22个氨基酸的活性肽,该活性肽具有Hepcidin基因家族特有8个半胱氨酸的保守序列.罗非鱼的Hepcidin基因结构包含3个外显子和2个内含子.本研究为今后阐明Hepcidin基因表达特性、表达产物理化性质、抗菌活性及其相关功能等奠定基础.     

Type IV collagens regulate BMP signalling in Drosophila

Dorsal-ventral patterning in vertebrate and invertebrate embryos is mediated by a conserved system of secreted proteins that establishes a bone morphogenetic protein (BMP) gradient. Although the Drosophila embryonic Decapentaplegic (Dpp) gradient has served as a model to understand how morphogen gradients are established, no role for the extracellular matrix has been previously described. Here we show that type IV collagen extracellular matrix proteins bind Dpp and regulate its signalling in both the Drosophila embryo and ovary. We provide evidence that the interaction between Dpp and type IV collagen augments Dpp signalling in the embryo by promoting gradient formation, yet it restricts the signalling range in the ovary through sequestration of the Dpp ligand. Together, these results identify a critical function of type IV collagens in modulating Dpp in the extracellular space during Drosophila development. On the basis of our findings that human type IV collagen binds BMP4, we predict that this role of type IV collagens will be conserved.     

薄壳山核桃原花青素合成关键酶基因的克隆与表达分析

【目的】原花青素是广泛存在于植物中一种重要的次级代谢产物,其强抗氧化性增强了植物自身的抗逆能力,同时赋予植物清除人体自由基的保健作用。研究薄壳山核桃种仁中原花青素生物合成途径,对改良薄壳山核桃的种质与品质均具有重要意义。【方法】以薄壳山核桃‘波尼’115和135 d种仁混合样品为材料,通过RT-PCR扩增、克隆和测序后,获得了薄壳山核桃原花青素合成关键酶相关基因CiDFR、CiLAR和CiANR的基因序列,并进行了生物信息学分析和表达水平分析。【结果】CiDFR基因长1 148 bp,包含1 020 bp的开放阅读框(ORF),编码339个氨基酸;CiLAR基因长1 390 bp,包含1 050 bp的ORF,编码349个氨基酸;CiANR基因长度为1 104 bp,包含1 014 bp ORF,编码337个氨基酸。荧光定量PCR检测结果显示,CiDFR和CiANR基因在种仁发育中期(95～105 d)表达量较高,之后快速下降至较低值;CiLAR基因在95 d表达量较高,之后快速降低,在155 d样品中表达量又升至最高点。【结论】CiDFR和CiANR基因表达量与酚类代谢物含量变化相关...     

野桑蚕漆酶基因克隆、序列分析及转录表达谱研究

利用RT-PCR方法,克隆了野桑蚕Bombyx mandarina漆酶基因,获得了其cDNA序列.该序列长2 317bp,含有一个2 295bp的完整开放阅读框,有8个外显子,7个内含子,编码一个由764个氨基酸残基组成的蛋白质,其蛋白质的分子量和等电点分别为84 340.91和6.61.推导的氨基酸序列与其它鳞翅目昆虫(Laccase)基因相应氨基酸序列有较高的同源性,该序列具有它们的漆酶基因所共有的典型特征.组织特异性表达分析表明了该基因仅在野桑蚕的表皮、头部、中肠和血液中有表达.这些结果为进一步研究野桑蚕漆酶基因的功能提供了分子基础.     

Cloning and expression pattern of a dehydrin-likeBDN1 gene from drought-tolerantBoea crassifolia Hemsl.

A 500-bp cDNA fragment was amplified via RT-PCR from drought-induced total RNA of the drought-tolerant B. crassifolia Hemsl. using primers based on the sequence of published dehydrin conserved region. By using 5′RACE, full-length coding region (1 148 bp) of BDN1 gene was produced. It is a new member of the dehydrin gene family. Southern analysis indicated that BDN1 is present in the B. crassifolia genome as a single-copy gene. Northern analysis revealed that its expression is inducible by drought and cold stresses as well as ABA application.     

点带石斑鱼鱼皮和鳞片胶原蛋白的提取及理化性质的研究

以点带石斑鱼为原料,分别从鱼皮和鱼鳞中提取酸溶性胶原蛋白,并研究其纯度、热变性温度、溶解度等理化性质.结果表明:(1)鱼皮和鱼鳞中胶原蛋白的提取率分别为76.21%和2.50%;(2)经紫外可见光谱扫描、傅里叶转换红外光谱和氨基酸分析,确定所提取的胶原蛋白是Ⅰ型胶原蛋白,具有完整的三螺旋结构;(3)SDS-PAGE电泳测定证明鱼皮和鱼鳞胶原蛋白均含有α1、α2、β和γ链,α1、α2链的相对分子质量分别为2.79×105和2.87×105,纯度分别为91%和85%;(4)鱼皮、鱼鳞胶原蛋白的热变性温度分别为40.16和42.06℃;(5)等电点分别为7.12和7.15;(6)两者在酸性pH(2～4)时具有高的溶解度,并且当NaCl的质量浓度分别为10和20 g/L时,溶解度最高.     

人类磷酸泛酰巯基乙胺结合蛋白新基因的分离和鉴定

在对AD293和HEK293进行差减杂交以探索两者在吸附和凋亡特性上的差异时,从AD293的高表达文库中分离得到一段新的cDNA片段.从人类胎脑文库克隆得到该基因,全长2 745 bp,编码的蛋白含518个氨基酸,被预测为磷酸泛酰巯基乙胺结合蛋白.该基因在染色体上定位于2p22.3,包含8个外显子.该cDNA编码的蛋白序列含有一个凋亡抑制蛋白5结构域,外皮蛋白重复片段和铜结合辛肽重复片段.RT-PCR分析显示该基因在人类正常组织和癌组织中广泛表达,但在癌组织中表达量相对较低,提示其可能对细胞凋亡有抑制作用.该基因在进化过程中高度保守.     

High cell density alters the ratio of type III to I collagen synthesis by fibroblasts

HUMAN fibroblasts in culture synthesise both type I and type III collagen(1), with type I accounting for 70-90% of the total(2). In culture, the rates at which these proteins are synthesised is constant and apparently rather rigidly controlled(3). However, the proportions of these collagens differs in cells cultured with increased amounts of serum (increased type III/I)(4) as well as in cells obtained from patients with certain diseases. Cells from patients with the Ehlers-Danlos type IV syndrome make little or no type III collagen(5,6), whereas cells from patients with osteogenesis imperfecta have an increased type III/I (refs 7, 8). We have found that cells from some patients with systemic sclerosis (scleroderma), have a reduced type III/I ratio. However, as previously reported, these cells grew to a lower density than control cells(9). We report here that normal fibroblasts from human and guinea pig skin produce proportionally more type III collagen at high cell density, probably because of a reduction in the synthesis of type I collagen.     

Critical test of a sister chromatid exchange model for the immunoglobulin heavy-chain class switch

B lymphocytes may switch from producing an immunoglobulin heavy chain of the mu class to that of the gamma, epsilon or alpha class. To maintain the specificity, the new heavy chain must keep the original variable (V) region; this is achieved by deleting DNA sequences so that the V (consisting of joined VH, diversity (DH) and joining (JH) gene segments) and C (constant) gene segments coding for the new heavy chain are brought into close proximity (reviewed in ref. 5; we do not consider here the mu-delta situation). There are, in principle, three types of chromosomal rearrangements that yield a deletion: rearrangement within a chromatid; unequal sister chromatid exchange (as suggested by Obata et al.); and unequal recombination between chromosomal homologues. We have analysed the arrangement of C mu DNA in clones of the pre-B-cell line 18-81 that switches in vitro from mu to gamma 2b. The clones examined produce either mu, gamma 2b or no immunoglobulin chain. We report here that all the gamma 2b clones had lost at least one copy of C mu and no clones contained three copies of C mu. These findings formally exclude both unequal sister chromatid exchange and recombination between homologues as mechanisms for creating a gene encoding the gamma 2b chain.