核糖体RNA拓扑学与RNAN－糖苷酶研究进展（上）

核糖体ＲＮＡ拓扑学的研究对阐明核糖体ＲＮＡ（ｒＲＮＡ）在蛋白质生物合成中的作用具有重要的意义。ＲＮＡＮ－糖苷０酶是一类核糖体失活蛋白．它只水解ｒＲＮＡ特定位置上一个腺苷酸的糖苷键,释放一个腺嘌呤碱基,使核糖体失活。ＲｉｃｉｎＡ链是研究得最早和最详细的ＲＮＡＮ－糖苷酶,迄今已发现有二十五种核糖体失活蛋白具有ＲＮＡＮ－糖苷酶活性。ＲＮＡＮ－糖苷酶作用于２８ＳｒＲＮＡ的α－ｓａｒｃｉｎ结构域,改变核糖体的构象而使其失活。     

RNA在翻译中的功能

ＲＮＡ在翻译中的功能关键词ＲＮＡ,翻译ｍＲＮＡ翻译为蛋白质需要氨酰ｔＲＮＡ与ｍＲＮＡ上的相应密码子相互作用。密码子和反密码子间的Ｗａｔｓｏｎ－Ｃｒｉｃｋ碱基配对的稳定性不足以保证有效、准确的解码,而要有核糖体的帮助。这种功能是由核糖体蛋白还是核糖体Ｒ...     

核糖体失活蛋白及核糖体拓扑结构的研究进展

天花粉毒蛋白使核糖体失活的分子机制是它有ＲＮＡＮ－糖苷酶的作用。从樟树种子中纯化的两种新的核糖体失活蛋白（ＲＩＰ）——辛纳毒蛋白和克木毒蛋白也都具有ＲＮＡＮ－糖苷酶和依赖超螺旋结构的核酸内切酶活性。辛纳毒蛋白还有杀虫活性;克木毒蛋白还有超氧化物歧化酶活性。被ＲＮＡＮ－糖苷酶失活的核糖体用硼氢化钠还原或氨基酸加成反应可部分地复活,这表明失活的核糖体ＲＮＡ上产生的一个活泼醛基对其失活起着重要作用。工作中建立了荧光标记在凝胶上测定小分子ＲＮＡ序列和定性测定糖蛋白的两种新方法。     

核糖体蛋白的核糖体外功能

核糖体蛋白的核糖体外功能关键词核糖体蛋白功能近年来发现,在核糖体蛋白中存在着可与ＤＮＡ结合的特征结构。人们由此推测,由核糖核酸（ＲＮＡ）转变为核蛋白体的核糖体可能是在ＲＮＡ上添加一些本来就存在的蛋白后形成的。人们还观察到,多种核糖体蛋白除组成核糖体外...     

核糖体失活蛋白及核糖体拓扑结构的研究进展（续完）

核糖体失活蛋白及核糖体拓扑结构的研究进展（续完）李向东刘望夷（中国科学院上海生物化学研究所,上海２０００３１）关键词核糖体失活蛋白核糖体拓扑结构ＲＮＡＮ－糖苷酶２．核糖体拓扑结构的研究核糖体是由数十种生物大分子（ＲＮＡ和蛋白质）构成的。早期普遍接受的...     

核仁小分子RNA

核仁小分子ＲＮＡ屈良鹄（中山大学生命科学学院生物工程研究中心广州５１０２７５）关键词核仁小分子ＲＮＡ（ｓｏｎＲＮＡ）;内含子（ｎｉｔｒｏｎ）;基因（ｇｅｎｅ）;核糖体（ｒｉｂｏｓｏｍｅ）真核生物（ｅｕｋａｒｙｏｔｅ）在真核生物细胞中,除了人们所熟悉的ｒＲＮＡ,ｍＲＮＡ和ｔＲＮＡ之外,还存在着许多小分子ＲＮＡ［１,２］,如核小分子ＲＮＡ（ｓｎＲＮＡ）,核仁小分子ＲＮＡ（ｓｎｏＲＮ）以及细胞过程中的调控活动,具有十分重要的生物学意义[3,4].     

揭示核糖体结构的秘密

近年来有科学家利用Ｘ射线晶体学、电子显微镜以及电子计算机绘制而成了核糖体的高分辨率图谱。该图谱揭示出一种细菌核糖体的 2个亚基的主要组成部分中的 2个ＲＡＮ分子及 31个蛋白质的大多数结构。耶鲁大学的ＰｅｔｅｒＢ .Ｍｏｒｒｅ在 2 0 0 0年 8月 11日的Ｓｃｉｅｎｃｅ上报道 ,他们从核糖体晶体中发现有一种非预料中的蛋白质包围了一个预料中的卷曲的ＲＮＡ。这种蛋白质经常有一个球状部分 ,另外还有一根短链延伸深入到核糖体结构中去。研究者将一种新分子导入核糖体 ,这种新分子停靠在肽键形成部位 ,由此他们进一步证实了位于隧道结…     

核糖体灭活蛋白在植物中的作用

植物核糖体灭活蛋白 (ribosome -inactivatingproteins ,RIPs)能够破坏真核或原核细胞的核糖体大亚基RNA ,使核糖体失活而不能与蛋白质合成过程中的延伸因子相结合 ,从而导致蛋白质合成受到抑制。不同的核糖体对不同RIPs的敏感性不同 ,RIPs对自体或异体核糖体的作用也有很大区别。RIPs对病毒有很强的抑制作用 ,并且有些RIPs表现出对某些真菌和昆虫的抗性 ,因此认为核糖体灭活蛋白在植物的防御反应中扮演重要角色。另外 ,RIPs还可能参与了细胞代谢、细胞死亡等生理调控过程。     

分子遗传学简介

rRNA （核糖体RNA) rRNA与蛋白质组成核糖体,核糖体是蛋白质合成 的场所。每个核糖体由大小两个亚基组成,它们的形 状、大小和化学组成是不同的。来源于真核细胞的核 糖体是由40S小亚基与60S大亚基组成的,它自身沉 降系数为80S。来源于原核细胞的核糖体则是由30S 小亚基与50S大亚基组成的,它自身沉降系数为70So     

酿酒酵母中的RNA解旋酶

ＲＮＡ解旋酶是一类能解开双链ＲＮＡ的酶 ,存在于所有生物体中 ,其家族成员在进化上具有保守序列。ＲＮＡ解旋酶属于分子伴侣 ,对于确保ＲＮＡ分子的正确折叠以及保持和修饰其特定的二级、三级结构必不可少。在核转录、前体ｍＲＮＡ剪接、核糖体生物发生、核质转运、翻译、ＲＮＡ降解以及结构基因表达等过程中ＲＮＡ解旋酶都发挥了一定的作用。1 .解旋酶的结构ＲＮＡ解旋酶大部分属于蛋白质超家族Ⅱ (ＳＦⅡ ) ,包含七个保守序列 (图 1 ) [1] 。除核心区域外 ,多数ＲＮＡ解旋酶具有不同的Ｎ末端和Ｃ末端部分 ,可能与底物专一性有关。目前 ,…     

Sequence homologies in the protamine gene family of rainbow trout

We have sequenced five different rainbow trout protamine genes plus their flanking regions. The genes are not clustered and do not contain intervening sequences. There is an extremely high degree of sequence conservation in the coding and 3' untranslated regions of the gene. Downstream sequences exhibit little homology though conserved regions are found 250 base pairs 3' to the gene. There are four regions upstream of the gene that are highly conserved in the six clones, including the canonical Goldberg - Hogness box which is 45 base pairs 5' to the coding region. A second homologous region is found 90 bases upstream. Although in the same approximate location as the CAAT box found upstream of other genes, it does not contain the canonical CAAT sequence. Further upstream of the protamine genes at -115 there is an A-T rich sequence while a 25 base pair conserved sequence is located 150 bases upstream. In addition we report the presence of a potential Z-DNA region of predominantly A-C repeats approximately one kilobase downstream of one of the genes.     

Polymorphism and expression of isoflavone synthase genes from soybean cultivars

Isoflavones are synthesized by isoflavone synthases via the phenylpropanoid pathway in legumes. We have cloned two isoflavone synthase genes, IFS1 and IFS2, from a total of 18 soybean cultivars. The amino acid residues of the proteins that differed between cultivars were dispersed over the entire coding region. However, amino acid sequence variation did not occur in conserved domains such as the ERR triad region, except that one conserved amino acid was changed in the IFS2 protein of the GS12 cultivar (R374G) and the IFS1 proteins of the 99M06 and Soja99s65 cultivars (A109T, F105I). In three cultivars (99M06, 99M116, and Simheukpi), most of amino acid changes were such that the difference between the amino acid sequences of IFS1 and IFS2 was reduced. The expression profiles of three enzymes that convert naringenin to the isoflavone, genistein, chalcone isomerase (CHI), isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) were examined. In general, IFS mRNA was more abundant in etiolated seedlings than mature plants whereas the levels of CHI and F3H mRNAs were similar in the two stages. During seed development, IFS was expressed a little later than CHI and F3H but expression of these three genes was barely detectable, if at all, during later seed hardening. In addition, we found that the levels of CHI, F3H, and IFS mRNAs were under circadian control. We also showed that IFS was induced by wounding and by application of methyl jasmonate to etiolated soybean seedlings.     

Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.     

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

Mammal-fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.     

Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb

Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog (Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants, ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Furthermore, a transgenic reporter gene flanked with this conserved non-coding region shows normal polarized expression in mouse limb buds. This conserved sequence has therefore been proposed to act as a long-range, cis-acting regulator of limb-specific Shh expression. Previous phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins. In this study, we first show that this conserved sequence motif is also physically linked to the Shh coding sequence in a teleost fish, the medaka, by homology search of a newly available genomic sequence database. Next, we show that deletion of this conserved intronic sequence by targeted mutation in the mouse results in a complete loss of Shh expression in the limb bud and degeneration of skeletal elements distal to the stylopod/zygopod junction. This sequence contains a major limb-specific Shh enhancer that is necessary for distal limb development. These results suggest that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control fin and limb development.     

Characterization of a cDNA clone coding for a sea urchin histone H2A variant related to the H2A.F/Z histone protein in vertebrates.

A cDNA clone coding for a sea urchin histone H2A variant has been isolated. The coding region of the clone has been sequenced and the sequence found to be closely related to the H2A.F sequence in chickens. The nucleotide sequence of the sea urchin H2A.F/Z is 74% conserved when compared to chicken H2A.F and 51% conserved compared to sea urchin H2A early and 60% compared to sea urchin H2A late. The nucleotide-derived amino acid comparisons show that H2A.F/Z is 97% homologous with H2A.F in chickens and 57% and 56% homologous when compared to sea urchin H2A early and late respectively. There are between 3-6 copies of the H2A.F/Z sequence in the S. purpuratus genome. The H2A.F/Z gene sequence codes for the previously identified H2A.Z protein. All embryonic stages and adult tissues tested contain mRNA for H2A.F/Z. The mRNA appears in the poly A+ RNA fraction after chromatography over oligo dT cellulose.     

Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli

A DNA sequence of 532 base pairs encompassing the entire Morganella morganii lipoprotein gene (lpp) was determined. Sequence comparisons of the M. morganii lpp gene with the lpp genes from Escherichia coli, Serratia marcescens, and Erwinia amylovora reveal that the M. morganii lpp gene is more distantly related to the E. coli lpp gene than any of the other lpp genes examined. Between the E. coli and M. morganii lpp genes, the following homologies were found: 44% in the promoter region (bases, -45 to -1), 88% in the 5'-end untranslated region of the mRNA, 58% in the signal sequence coding region, 75% in the coding region for the first 51 and 43% for the last 7 amino acid residues. Upstream of the promoter region and downstream of the termination codon, there are extensive insertions, deletions, and base substitutions. In spite of the differences in the DNA sequences, the lipoprotein structure was found to be highly conserved except for the carboxyl-terminal sequence of 7 amino residues. The coding region of the M. morganii lpp gene including the signal sequence was inserted into an expression cloning vector so that the production of the M. morganii lipoprotein could be induced in E. coli by a lac inducer, isopropyl-beta-D-thioglactoside. It was found that when induced, the M. morganii prolipoprotein was apparently secreted normally across the E. coli cytoplasmic membrane, modified with glycerol and palmitic acid, processed to the mature lipoprotein, and assembled in the E. coli outer membrane. The bound form covalently linked to the peptidoglycan was also found.     

Molecular cloning and sequencing of a cDNA coding for mature human kidney cathepsin H

A cDNA library was established from human kidney RNA and screened with an extended oligonucleotide probe derived from the amino-acid sequence of human cathepsin H. A recombinant clone, pRF15, was isolated and characterized. DNA sequence analysis of its 1106-nucleotide-long insert revealed that pRF15 encodes the complete protein sequence of mature cathepsin H plus 28 amino acids of a propeptide, thus confirming that cathepsin H is synthesized as a larger precursor molecule and posttranslationally processed. Northern blot analysis indicated that cathepsin H is predominantly synthesized in kidney. A high degree of sequence homology was observed with rat cathepsin H, especially within the propeptide. The part of the prosequence coding for the "minichain" is conserved in the prosequence of aleurain, a plant thiol protease.