酵母基因组DNA的两个简易制备方法

酵母基因组ＤＮＡ的制备一般需要制作原生质体。我们基于丝状真菌的方法[1,2 ] 发展了 2个酵母基因组ＤＮＡ的制备程序 ,取得了良好的效果。1　材料与方法1.1　材料菌种　野生酿酒酵母菌种Ｇ1Ｍ 2 34为本中心周惠副教授惠赠。1.2　方法1.2 .1　酵母基因组ＤＮＡ制备方法　①方法 1:接种酵母菌种于无菌的 5 0ｍＬＹＰＤ或ＳＤ培养基 ,在30℃生长至对数生长晚期。滤液 4 0 0 0ｒ/ｍｉｎ离心10ｍｉｎ。菌体用液氮碾磨破壁。加 7ｍＬＤＮＡ缓冲液 (10 0ｍｍｏｌ/ＬＴｒｉｓ ＨＣｌ,ｐＨ 8.0 ,10ｍｍｏｌ/ＬＥＤ ＴＡ ,1%ＳＤＳ)。混匀 ,6 5℃保…     

一种新的DNA分子克隆方法

DNA分子克隆是基本的分子生物学实验技术,传统的分子克隆方法大多需经过酶切链接过程,但在某些情况下,没有合适的酶切位点往往会成为阻碍克隆进行的障碍.本文描述了一种新的分子克隆方法,称为不依赖酶切和链接的分子克隆（RLIC）.利用RLIC,将3种不同大小的DNA片段克隆到3种不同载体,证明了这种方法的有效性和可靠性.由于该方法不受限制性酶切序列限制,省去了酶切连接步骤,因此具有很大的灵活性和简便性,在分子生物学研究方面有广泛应用前景.     

tRNA衍生片段和tRNA半分子的生物学功能及其在疾病发生中的作用

tRNA衍生片段(tRNA-derived RNA fragment,t RF)和tRNA半分子(tRNA halves,ti RNA)由成熟tRNA或其前体tRNA在不同位点特异性剪切产生,它们是一类广泛存在于原核生物和真核生物转录组中的非编码小RNA分子.t RF主要有tRF-5、tRF-3和tRF-1等3亚类,分别来自成熟tRNA的D环至反密码环茎区间切割至5′端、T环开始至3′端和前体tRNA的3′端尾部,其长度为14~30个核苷酸(nucleotide,nt).ti RNA主要有5′ti RNA和3′ti RNA等2亚类,是在成熟tRNA反密码子环处切割分别产生,其长度为29~50 nt.t RF和ti RNA具有多种生物学功能,既可以在应激反应中作为信号分子,又可以作为基因表达的调节者.它们与人类多种疾病(如肿瘤、神经退行性疾病、代谢性疾病和传染病等)的发生密切相关,有希望成为疾病诊断的新型标志物.本文就t RF和ti RNA的分类、生物学功能以及与人类疾病的关系作一综述.     

水稻核基因组DNA的YAC克隆和鉴定

将EcoRI部分消化的水稻(Oryza sativa L.)细胞核高分子量DNA电泳分部,回收大于200kb的片段,与内切酶消化过的酵母人工染色体(YAC)双质粒载体pJs97。pJS98DNA连接,转化酵母YPH252感受态原生质球,用ura^-,TrP^-双选择培养基直接筛选转化子,已获得2 000多个克隆。转化子DNA的southern杂交显示插入片段在200～820kb范围。     

克隆DNA的定向诱变

克隆ＤＮＡ的定向诱变史桂荣（黑龙江省农科院原子能利用研究所,哈尔滨）基因诱变技术是遗传学研究中最重要的手段之一。随着分子遗传学的发展,人们开始按照自己的意愿有目的地在离体条件下创造基因突变,然后把它置入生物体基因组中,观察分析这些突变的表型效应。这种...     

酵母丙氨酸tRNA的纯化、半分子制备及其末端鉴定

本文报道了用DEAE-葡聚糖凝胶A-25柱层析纯化酵母丙氨酸tRNA 的方法,纯化的tRNA,按接受丙氨酸的活力计算,其纯度达60～70%。经核糖核酸酶T_1(RNase T_1)限制性降解(tRNA 与RNase T_1的比率为1毫克/15单位,0℃,4分钟),柱层析,制备了tRNA 的两个半分子。用萤光标记法,[~3H]标记法,测得5′半分子的3′末端为鸟苷酸;用[~(32)P]标记法测得3′半分子的5′末端为胞苷酸。因此,RNase T_1限制性降解丙氨酸tRNA 的切点在反密码子的G—C 键之间。     

酵母丙氨酸tRNA的纯化、半分子制备及其末端鉴定

本文报道了用DEAE-葡聚糖凝胶A-25柱层析纯化酵母丙氨酸tRNA的方法,纯化的tRNA,按接受丙氨酸的活力计算,其纯度达60～70%。经核糖核酸酶T_1(RNase T_1)限制性降解(tRNA与RNase T_1的比率为1毫克/15单位,0℃,4分钟),柱层析,制备了tRNA的两个半分子。用萤光标记法,[~3H]标记法,测得5′半分子的3′末端为鸟苷酸;用[~(32)P]标记法测得3′半分子的5′末端为胞苷酸。因此,RNase T_1限制性降解丙氨酸tRNA的切点在反密码子的G—C键之间。     

酶母K.cicerisporus基因组中有启动子功能的DNA片段的克隆

利用以３′－氨基糖苷磷酸转移酶基因（ＡＰＨＩ）为报道基因的一套启动子探针质粒ｐＳＫ－ｋａｎ４０１、ｐＳＫ－ｋａｎ１１０５、ｐＳＫ－ｋａｎ１２３８,从酵母Ｋｌｙｖｅｒｏｍｙｃｅｓ ｃｉｃｅｒｉｓｐｏｒｕｓ基因组中克隆到８个有较强启动功能的ＤＮＡ片段,分析出３′端ＤＮＡ序列,并在酵母Ｋｌｕｙｖｅｒｏ８ｍｙｃｅｓｅ ｌａｃｔｉｓ中通过检测报告基因与３－磷酸甘油醛脱氢酶基因（ＡＰＤＨ）的ｍＲＮＡ表达量的比     

Gene Cloning, Overproduction and Purification of Escherichia coli tRNA_2~(Arg)

合成的编码大肠杆菌ｔＲＮＡＡｒｇ２的基因,嵌入受ＩＰＴＧ诱导启动子控制的质粒ｐＴｒｃ９９Ｂ中。用上述含目的基因的质粒转化大肠杆菌ＭＴ１０２,得到ｔＲＮＡＡｒｇ２基因序列正确的克隆。诱导表达后,与受体菌相比,转化子中的ｔＲＮＡＡｒｇ的含量高出１０倍,ｔＲＮＡＡｒｇ２的含量高出３０倍,占总ｔＲＮＡ的７０％。ＤＥＡＥＳｅｐｈａｃｅｌ柱层析后,ｔＲＮＡＡｒｇ２的纯度即可达到８８％。再用ｂｅｎｚｙｌＤＥＡＥｃｅｌｕｌｏｓｅ柱层析可得到纯度为９９％、精氨酸接受活力为１６００ｐｍｏｌｅ／Ａ２６０单位的ｔＲＮＡＡｒｇ２。从４升过夜培养液中得到的４０ｍｇ总ｔＲＮＡ。从中可得到１８ｍｇ纯ｔＲＮＡＡｒｇ２,产率为６２％。首次精确地测定了精氨酰ｔＲＮＡ合成酶催化ｔＲＮＡＡｒｇ２时的动力学常数。     

Trm112p Is a 15-kDa Zinc Finger Protein Essential for the Activity of Two tRNA and One Protein Methyltransferases in Yeast

The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 (mcm⁵U₃₄ and mcm⁵s²U₃₄), the formation of which requires at least 25 different proteins. The addition of the last methyl group is catalyzed by the methyltransferase Trm9p. Trm9p interacts with Trm112p, a 15-kDa protein with a zinc finger domain. Trm112p is essential for the activity of Trm11p, another tRNA methyltransferase, and for Mtq2p, an enzyme that methylates the translation termination factor eRF1/Sup45. Here, we report that Trm112p is required in vivo for the formation of mcm⁵U₃₄ and mcm⁵s²U₃₄. When produced in Escherichia coli, Trm112p forms a complex with Trm9p, which renders the latter soluble. This recombinant complex catalyzes the formation of mcm⁵U₃₄ on tRNA in vitro but not mcm⁵s²U₃₄. An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell.     

S1核酸酶作用与微环DNA分子的克隆策略

米曲霉来源的S1 核酸酶具有降解单链DNA或RNA的作用。在适当的条件下 ,该酶能将不同的环形DNA分子从超螺旋转变成开环和线形结构 ,对质粒pUC19的实验证明 ,S1 核酸酶的这种转变作用与加入的酶量呈正相关。在 2 5 μL总反应体积中 ,按 10 0ngDNA加入 5u至 17u的S1 核酸酶 ,能获得较高比例的线形DNA。由于微环DNA分子太小 ,单酶切位点的出现率较低 ,很难用常规方式进行克隆 ,以S1 核酸酶进行线形化是微环DNA克隆的途径。pC3是已知最小的真核生物线粒体DNA类质粒 (5 37bp) ,经S1 核酸酶线形化后 ,成功地克隆到pMD18 T载体上。     

Chemical Incorporation of 1-Methyladenosine,Minor tRNA Component,into Oligonucleotides

Abstract

The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.     

A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast

Summary Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA _I ^Met . Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA _I ^Met is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slowly; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.Abbreviations IMT and imt (imt=initiator methionine tRNA), designate the genotype of the wild-type and the mutant alleles respectively, of the initiator methionine transfer RNA gene - met-tRNA _I ^Met methionylated initiator methionine transfer RNA - eIF-2 eukaryotic initiation factor two - GTP guanosine 5-triphosphate The calculation of Td values (the temperature at which half of the duplex is dissociated) for oligonucleotides used as probes in hybridizations was based on the assumption that the increase in Td value was 4° C for each G:C base pair and 2° C for each A:T base pair (Wallace et al. 1981)     

The origin of the tRNA molecule: implications for the origin of protein synthesis

A model for tRNA molecule origin is discussed. The model postulates that this molecule originated simply by direct duplication (and subsequent evolution) of a gene coding for an RNA hairpin structure, which can thus be hypothesized as the evolutionary precursor of the tRNA molecule. The main properties are defined for these hairpin structures and it is suggested that these structures might have housed, near their 3' end, anticodons that were transferred to the loop of the tRNA anticodon during duplication of the hairpin structures. Moreover, the main characteristics are given for the evolutionary intermediary formed by direct duplication of the hairpin structure, i.e. the double hairpin. The evolutionary stages envisaged by this model for tRNA origin seem to naturally imply some evolutionary transitions through which the origin of protein synthesis passed. Finally, some strong historical evidence is provided to corroborate the model.     

Patterns of evolution in the unique tRNA gene arrays of the genus Entamoeba

Genome sequencing of the protistan parasite Entamoeba histolytica HM-1:IMSS revealed that almost all the tRNA genes are organized into tandem arrays that make up over 10% of the genome. The 25 distinct array units contain up to 5 tRNA genes each and some also encode the 5S RNA. Between adjacent genes in array units are complex short tandem repeats (STRs) resembling microsatellites. To investigate the origins and evolution of this unique gene organization, we have undertaken a genome survey to determine the array unit organization in 4 other species of Entamoeba-Entamoeba dispar, Entamoeba moshkovskii, Entamoeba terrapinae, and Entamoeba invadens-and have explored the STR structure in other isolates of E. histolytica. The genome surveys revealed that E. dispar has the same array unit organization as E. histolytica, including the presence and numerical variation of STRs between adjacent genes. However, the individual repeat sequences are completely different to those in E. histolytica. All other species of Entamoeba studied also have tandem arrays of clustered tRNA genes, but the gene composition of the array units often differs from that in E. histolytica/E. dispar. None of the other species' arrays exhibit the complex STRs between adjacent genes although simple tandem duplications are occasionally seen. The degree of similarity in organization reflects the phylogenetic relationships among the species studied. Within individual isolates of E. histolytica most copies of the array unit are uniform in sequence with only minor variation in the number and organization of the STRs. Between isolates, however, substantial differences in STR number and organization can exist although the individual repeat sequences tend to be conserved. The origin of this unique gene organization in the genus Entamoeba clearly predates the common ancestor of the species investigated to date and their function remains unclear.     

K562细胞中锌指蛋白cDNA基因片段的克隆

 Ｋ５６２细胞中锌指蛋白ｃＤＮＡ基因片段的克隆刘智,朱定尔,谢慎思,朱小湘,肖广惠,陈汉春（湖南医科大学分子生物学研究室,长沙４１００７８）１９８５年,Ｍｉｌｌｅｒ等［１］等分离并测定了非洲爪蟾卵母细胞转录因子ⅢＡ（ＴＦⅢＡ）的ｃＤＮＡ序列,推出蛋白质...