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1.
在大肠杆菌中克隆肺炎支原体P1蛋白羧基端基因片段,为P1蛋白基因片段的扩增、表达及探讨羧基端基因片段功能打基础.采用PCR扩增方法获取P1结构基因.扩增产物用SalI和EcoRI酶切消化,回收1kb大小的DNA片段并与pUC19DNA连接,转入大肠杆菌JM109菌株.用X-gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定.经PCR扩增MPDNA获得1条5.0kbDNA片段.重组质粒限制性内切酶指纹图谱显示出2条带,1条为pUC19载体DNA带,另1条是1kb的插入片段.实验获得肺炎支原体P1蛋白结构基因及含P1蛋白羧基端DNA片段的重组克隆株.  相似文献   

2.
 本文采用限制内切酶HindⅢ切割经Sepharose 4 B柱纯化的北京鸭肝线粒体DNA,得到五个片段,其大小分别为:A——6.56kb,B——3.12kb,C——3.12kb,D——2.40kb,E——1.40kb。以质粒pWR33为载体,在HindⅢ切点处插入线粒体DNA的HindⅢ酶切片段,将体外重组质粒转化到大肠杆菌HB101内,经筛选、分析:如菌落原位杂交,限制性内切分析和Southern吸印法分析,首次得到了线粒体DNA的HindⅢB、C、D、E四个片段的克隆子。另外还得到了一个与片段A同源的1.60kb大小的片段。  相似文献   

3.
用重组DNA技术构建了病毒ki-ras表达质粒,用Eco R Ⅰ和Bam H Ⅰ酶解pUC-9DNA,小牛肠碱性磷酸酶脱磷酸,病毒ki-ras编码基因来源于Hi-Hi-3质粒DNA的Sat Ⅱ和Eco R Ⅰ酶切的0.6kb片段,用连接酶和DNA聚合酶Klenow片段将两个片段连接,转化大肠杆菌JM103,免疫筛选表达质粒,从156个转化克隆中得到两株表达质粒,其中一株pKras83的p21蛋白表达量为细菌总蛋白量的10%。  相似文献   

4.
高质量粘粒基因组文库构建的关键是HMW DNA的长度至少为粘粒载体容量的10倍,通常粘粒载体的容量为30~50 kb,因此提取的HMW DNA应不小于500 kb.HMW DNA在制备时不能受到任何物理的剪切力,以免DNA断裂和损伤.利用琼脂糖凝胶包埋制备的DNA胶块经裂解和纯化后发现其DNA长度远大于500 kb,明显优于商品化试剂盒提取和酚抽提法.用BamHⅠ、Sau3AⅠ、XbaⅠ和HindⅢ对DNA胶块进行不完全酶切研究表明,构建文库常用的Sau3AⅠ并不适合胶块内酶切反应,而BamHⅠ酶切B.cepacia HMW-DNA效果较好,产生的DNA片段集中在20~50 kb之间,完全适合粘粒基因组文库的构建,为B.cepacia大插入片段基因组文库的构建以及功能基因组的研究奠定了良好的理论基础.  相似文献   

5.
蚕豆叶绿体DNA(ct—DNA)经BamH I酶切产生26个片段,最大的为14.00kb,最小的为0.42kb。本文以pBR322为载体,E.Coli HB101为受体菌,采用标准分子克隆法构建了蚕豆ct—DNA BamH I克隆库,并从库中分离得到含叶绿体rRNA基因的克隆。32P标记的E.Coil 16S、23S rRNA能和蚕豆ct—DNA BamH I第6(B6,5.65kb)和第9(B9,4.70kb)个片段杂交,含有这二个片段的克隆分别命名为pVFB32和pVFBl6。利用几种限制性内切酶酶切和Southern印迹法构建了pVFBl6的物理图谱。pVFBl6电镜下观察到有一变性环(A—T丰富区),经Hind I酶切,电镜观察定位此A—T丰富区位于16S和23S rRNA基因的间隔顺序内,推测该环可能与DNA复制有关。  相似文献   

6.
一种新型可用于DNA分子量标准的质粒构建   总被引:1,自引:1,他引:0  
将首尾带有EcoR Ⅰ酶切位点的2.0kb、1.5kb、1.0kb、0.75kb、0.5kb、0.25kb六个长度的片段逐步连接到pGEM-3zf( )质粒上的B.am H I位点中,构建的质粒用EcoR Ⅰ进行单酶切,经电泳可以获得七条DNA带与设计结果完全一致,可用于DNA电泳试验中分子量标准。  相似文献   

7.
以 pBR322 DNA 为载体,Escherichia coli HB101为受体菌,克隆了含蚕豆叶绿体 rRNA基因的二个 BamHI 片段。应用几种限制性内切酶酶切以及 Southern 印迹法构建了这二个特异片段的物理图谱。重组质粒 pVFB16含有一个4.70kb 的 BamHI 片段,其上含有完整的16S rRNA 基因;重组质粒 pVFB32含有一个5.65kb 的 BamHI 片段,其上含有23S rRNA基因,23S—4.5S/5S rRNA 基因的间隔区及4.5S/5S rRNA 基因。  相似文献   

8.
水稻核基因组DNA的YAC克隆和鉴定   总被引:1,自引:0,他引:1  
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。  相似文献   

9.
萝卜叶绿体DNA花粉育性特异片段的定位   总被引:4,自引:0,他引:4  
本实验采用萝卜细胞质雄性不育系401A及其同核保持系401B为材料,利用BamH I、EcoR I、BglI和HindⅡ四种内切酶酶解其叶绿体:DNA。除HindⅡ外,其余三种内切酶电泳图谱中均显示两系之间的明显差异。将BamH I的5个差异片段分别与载体pBR322进行体外连接,获得4种重组体克隆。利用3种膜蛋白质基因探针,分别与两系的叶绿体DNA杂交,杂交均未出现在差异片段所在部位,这说明,两系之间的差别可能与这3种基因无关。将重组体质粒分别与P700叶绿素a脱辅基蛋白质基因探针及反向重复区内rRNA基因区探针杂交,结果表明,有3种重组体质粒所携带的差异片段与rRNA基因探针在杂交中显示出明显的阳性反应。也就是说,这3个差异片段均位于rRNA基因所在的叶绿体基因组的反向重复区中。  相似文献   

10.
油菜细胞质雄性不育系叶绿体DNA特异片段的分子克隆   总被引:7,自引:0,他引:7  
采用高离子浓度缓冲液法分别提取油菜不育系及保持系的叶绿体DNA。经Sepharcse 4B柱层析纯化后,得到具有较高纯度的叶绿体DNA样品。将其分别用Eco RI、Bam HI、HimdHI、PstI和XhoI 5种限制性内切酶酶解,得到5种限制性内切酶图谱。其中除PstI图谱外,其它4种酶谱均显示出明显的两系间叶绿体DNA结构差异。以pBR 322为载体,分别克隆了不育系Bam HI图谱上的3个特异片段。得到的3个克隆,经克隆杂交及电泳分析后,证实分别带有上述3个目的片段。这些特异片段的特性及其与花粉育性的关系尚在研究中。  相似文献   

11.
Bulk DNA isolated from the ectomycorrhizal basidiomycete Hebeloma circinans was treated with proteinase K and submitted to agarose gel electrophoresis. In addition to high molecular weight genomic DNA, three minor bands were detected. The band with the highest electrophoretic mobility (2.2 kbp) corresponds to double-stranded RNA. The two other bands, termed pHC1 and pHC2, were shown to be dsDNA molecules of 10.3 and 9.1 kbp, respectively. Treatment of the pHC elements with 3'- and 5'-specific exonucleases revealed a linear structure and proved that the 5' ends are protected from digestion; for pHC2, linearity was confirmed by restriction mapping. A 3.2 kbp HindIII fragment of pHC2 was cloned and sequenced; it contains two open reading frames encoding putative viral B type DNA and RNA polymerases. Thus, the fungus harbors a typical linear plasmid, up to now, rarely described for basidiomycetes and hitherto unknown for mycorrhizal species.  相似文献   

12.
Extrachromosomal elements were found in a strain ofX. dendrorhous, and were characterized as linear DNA forming two well defined groups, pPh1 with 3 high-copy-number molecules, pPh11 (6.9 kb), pPh12 (5.7), pPh13 (4.7), and pPh2 with 2 low-copy-number molecules, pPh21 (3.6 kb), pPh22 (3.0). A 4077 bp fragment from pPh13 was cloned in pUC18 (pDK1) and sequenced (accession no. AJ 278 424). Seven putative ORF and some possible regulator sequences were defined.  相似文献   

13.
14.
Sun Y  Wei W  Ding X  Xia L  Yuan Z 《Archives of microbiology》2007,188(4):327-332
The association of 20 kb heterologous DNA fragments with the parasporal crystals from native and recombinant Bacillus thuringiensis strains was analyzed, respectively. The cry2Aa10 gene cloned in plasmid pHC39 was transformed into B. thuringiensis subsp. kurstaki strains CryˉB and HD73, producing recombinant strains CryˉB(pHC39) and HD73(pHC39). SDS-PAGE and scanning electron microscopy analyses demonstrated that the recombinant CryˉB(pHC39) produced cuboidal crystals of Cry2Aa10 protoxin, while recombinant HD73(pHC39) produced both bipyramidal crystals of Cry1Ac1 protoxin and cuboidal crystals of Cry2Aa10 protoxin. Bioassay results proved that recombinant HD73(pHC39) showed higher insecticidal activity to Helicoverpa armigera than CryˉB(pHC39). It was found that 20 kb DNA fragments were present in bipyramidal and cuboidal crystals from both native and recombinant strains, and the 20 kb heterologous DNAs contained chromosome-specific and resident large plasmid-borne DNA fragments, suggesting the 20 kb heterologous DNA fragment embodied in crystals came randomly from the bacterial chromosomal and plasmid genome. This was the first investigation devoted exclusively on the origin of 20 kb DNA fragments in the parasporal crystals of B. thuringiensis. The data provides a basis for further investigation of the origin of 20 kb DNAs in the crystals and the interaction of DNA and protoxins.  相似文献   

15.
A 323-bp DNA fragment (U15557) was isolated, cloned, and sequenced after polymerase chain reaction (PCR) amplification from Monodelphis domestica genomic DNA. AHindIII restriction fragment length polymorphism was identified in this species using the U15557 PCR. fragment as a hybridization probe. DNA samples exhibited either a 6.4kb band, a 7.2 kb band, or both bands simultaneously. Behaviour of these two variants in family studies was consistent with codominant autosomal inheritance. Linkage between this marker and the loci encoding protease inhibitor (PI) and adenylate kinase 1 (AK1) was found in M. domestica.  相似文献   

16.
Entamoeba histolytica genome was analysed by pulsed field gel electrophoresis under conditions to separate linear chromosomes in the 170–1400 kb range. We identified linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb (mean sizes obtained by three different methods) and we estimated their reorientation times and migration velocities at various experimental conditions. DNA shift mobility assays, using ethidium bromide, suggested that bands migrating at 227 and 631 kb contain linear and circular DNA, whereas a band at 436 kb has only circular DNA. We obtained a regression equation relating sizes of supercoiled DNA molecules with their migration velocities during a pulse at constant electric field and temperature. We also developed a computer program (EHPATTERNS) that predicts the migration per pulse and the resolution order of circular and linear E. histolytica DNA at different pulse times and constant driving and frictional forces. The simulation showed that linear DNA molecules frequently co-migrate with circular molecules, but circular molecules change when the pulse time varies. This molecular mixture generates broad bands and difficulties in the interpretation of the molecular karyotype of E. histolytica. Received: 19 January 1999 / Revised version: 3 November 1999 / Accepted: 22 November 1999  相似文献   

17.
Summary Certain physicochemical properties of rice mitochondrial DNA (mtDNA) were determined. Certain low-molecular-weight mtDNA bands were found in addition to the major mtDNA band. Rice mtDNA appeared in the electron microscope as a collection of linear molecules with heterogeneous length in the range of 1–156 kb. The major distribution area was 60–105 kb. A small fraction (less than 5%) of rice mtDNA was found in the form of a circular molecule. Some molecules had the appearance of being supercoiled. Replication fork structures were found in both circular and linear mtDNA molecules. In one rice species, Jin Nante, 15 different circular molecules were found. Rice mtDNA was digested with different restriction enzymes. The total molecular weight of rice mtDNA was calculated to be about 300 kb according to the data of restriction enzyme digestion and electron microscopy.  相似文献   

18.
19.
The accumulation of subgenomic phage φ29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb. Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage φ29 genome. The nature of the 4, 2.3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single-stranded self-complementary strands forming hairpin structures. The other molecules consisted of palindromic linear double-stranded DNA molecules. Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo . Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5'-TGTTtCAC-GTGgAACA-3' being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the φ29 genome. In addition, the almost identical sequence 5'-TgGTTTCAC-GTGGAAtCA-3' was found once. These five DSEs are all located in the right half of the φ29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene of the B. subtilis chromosome. Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE. As the same subgenomic φ29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.  相似文献   

20.
Summary This paper reports the discovery and initial characterization of two small plasmids, pCfl and pCf2, in the marine diatomCylindrotheca fusiformis. Extracted diatom DNA separates into two bands in CsCI-Hoechst 33258 dye gradients. Upon agarose gel electrophoresis of a sample of the upper band of the gradient we observed, in addition to high molecular weight (genomic) chloroplast and mitochondrial DNA, pairs of lower molecular weight bands. These bands contained two species of circular plasmid DNA molecules, as shown by electron microscopy. The nucleotide composition of the plasmids, and chloroplast and mitochondrial DNAs is similar, as indicated by their co-banding in the gradients. They were cloned, and their restriction maps determined, showing that pCfl is 4.27 and pCf2 4.08 kb in size. By hybridization analysis, we showed that pCfl and pCf2 share regions of similarity, but not identity. Neither plasmid hybridizes with mitochondrial DNA. Both plasmids hybridize with chloroplast DNA, and pCf2 also hybridizes with nuclear DNA.  相似文献   

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