大白菜细胞质雄性不育系RAPD特异扩增片段的克隆及其序列分析

利用RAPD技术从大白菜细胞质雄性不育系CMS3411-7的DNA中得到1个特异扩增片段CMSO-PL01670,进一步以该片段为探针进行Dot和Southern杂交证实了该片段为不育系所特有。回收该特异扩增片段并将其克隆到pGEM-T Easy载体上进行序列测定。测序结果表明该片段全长673bp,其碱基组成为A T=67.16％。通过与GenBank EMBL DDBJ PDB中的455 972个序列进行比较,同源性均小于30％,表明该片段为一新发现序列。并且该序列的 4 - 216的区域内含有1个可编码70个氨基酸的开放阅读框架。上述结果为今后从分子水平进一步研究大白菜细胞质雄性不育打下了坚实的基础。     

采用苯酚羟化酶基因特异引物检测苯酚降解菌

根据苯酚羟化酶基因高度保守序列设计了一对该基因的特异PCR引物。采用该特异引物从苯酚降解菌醋酸钙不动杆菌 (Acinetobactercalcoaceticus)PHEA 2的总DNA中扩增到唯一一条大小为 684bp的片段。该DNA片段与已知的A .calcoaceticusNCIB82 50的苯酚羟化酶基因具有高度的同源性 ,其核苷酸序列的同源性为 84% ,推导的氨基酸序列的同源性为 98%。对苯酚和非苯酚降解菌株的PCR扩增结果表明 :所有苯酚降解菌均能扩增出 684bp的特征片段 ,而非苯酚降解菌则无PCR条带。对炼焦废水中的细菌群落进行PCR扩增和生化特性检测表明 :显示 684bp特征片段的菌株均具有苯酚降解特性。上述结果表明 ,利用苯酚羟化酶基因的特异引物可对环境中的苯酚降解菌株进行准确快速的PCR检测。     

菠菜CBF转录因子基因片段的克隆及序列分析

根据拟南芥CBF基因序列的保守区设计合成一对特异引物,以菠菜基因组DNA为模板,采用PCR扩增的方法扩出一条DNA特异片段并克隆到pMD18-T载体中。用PCR法和酶切分析法对克隆片段进行鉴定并进一步进行核苷酸序列分析。序列测定该片段长为423bp。OMIGA2.0软件分析结果表明,该片段的推断氨基酸序列与黑麦(AAL35759)、小麦(AAL37944)、拟南芥(AAC78646)、大麦(AAL84170)的同源性分别为33.8%、33.1%、30.8%和30%。     

雄牛特异的SRY同源序列的扩增和分析

利用人、兔、鼠SRY序列设计引物,应用PCR扩增牛的SRY序列,获得200bp的雄牛特异的扩增片段。克隆该扩增片段,获得重组质粒pCH21,进行序列分析,并与人、兔和鼠SRY的对应区域比较,具有高度同源性。用pCH21 DNA作探针与牛的基因组DNA酶切图谱杂交,显示了雄牛特异的I．7kb的杂交带。分析200bp的PCR扩增片段是牛的SRY基因片段。用同一对引物扩增人和山羊的DNA样品,也获得雄性特异的200bp的扩增片段。     

白沙蒿肌动蛋白基因核心片段的克隆和序列分析

本研究以荒漠植物白沙蒿总RNA为模板,运用RT-PCR方法扩增出肌动蛋白基因核心序列.将获得的片段克隆到T载体后进行测序,序列分析表明:白沙蒿肌动蛋白基因核心片段长599 bp,编码198个氨基酸.将该序列在GenBank中注册,并与多种植物肌动蛋白序列进行同源性比较,发现该片段的核酸序列同源性在75%以上,氨基酸序列同源性在85%以上,具有高度保守性.     

毛柄金钱菌gpd-Fv启动子的克隆及序列分析

根据Genbank登陆的毛柄金钱菌gpd启动子序列设计引物,对毛柄金钱菌基因组DNA进行PCR扩增,扩增获得了2条特异片段gpd-Fv1和gpd-Fv2;回收特异片段后分别克隆进T-easy载体中并进行DNA序列测序,结果表明:gpd-Fv1和gpd-Fv2长度分别为1412 bp和752bp.通过同源性分析,结果表明:gpd-Fv1和gpd-Fv2的DNA序列与已报道的毛柄金钱菌gpd启动子(Genbank:AF515622.1)的同源性分别为95%和98%.进一步通过软件预测,结果表明克隆的2条片段具有基本的启动子结构,存在多个转录因子结合位点.根据启动子的序列特点,初步构建了毛柄金钱菌gpd启动子模型.     

蒙古牛BMPR-IB基因部分cDNA的克隆和序列分析

BMPR-IB基因主要在哺乳动物卵巢中表达,对卵泡的发育和分化起重要作用。该研究从影响卵巢生长发育和调控的BMPR-IB基因出发,以牛卵巢的RNA为模板,按照不同物种BMPR-IB基因的相似性设计特异引物,运用RT-PCR技术扩增并获得了特异片段,该片段经PCR、酶切和测序验证,证实所克隆序列为牛BMPR-IB序列,包含有953bp组成的部分cDNA序列,同源性分析结果表明,牛BMPR-IB基因序列与绵羊、山羊、人、猪、小鼠的BMPR-IB基因分别为98%、97%、92%、93%、88%的同源性。这为克隆其他物种的BMPR-IB基因提供了依据,同时牛骨形态发生蛋白的测序为更好地理解牛的生殖机理提供帮助。     

我国东北地区蜱中检出蜱传埃立克体DNA

应用16S rRNA基因构建的特异性引物进行半套式PCR,检测东北地区蜱标本中埃立克体DNA,并对扩增产物进行克隆和序列测定,与GenBank中已知序列进行同源性比较。结果从全沟硬蜱和森林革蜱中均扩增出了查菲埃立克体DNA,扩增片段与美国分离株(GenBankM73222)相对应片段完全一致,同源性为100%;另外从全沟硬蜱中扩增出了粒细胞埃立克体DNA,扩增片段与美国分离株(GenBankU02521)相对应片段相差2个碱基,同源性为99.7%。表明我国东北边境地区有埃立克体病的病原体存在,该地区可能存在埃立克体病的自然疫源地。     

用RACE技术扩增并克隆牛BMP4基因3'端序列

RACE技术是一项扩增基因末端序列的新技术。该研究从牛BMP4基因出发,以牛软骨的RNA为模板,按照不同物种BMP4基因的相似性设计特异引物,运用PCR和RACE技术扩增并获得了特异片段,该片段经PCR、酶切和测序验证,证实所克隆序列为牛BMP4的3′端序列,包含有1170bp组成的开放读码框(ORF),编码389个氨基酸,3′非编码区121bp个核苷酸和poly(A)15。同源性分析结果表明,牛BMP4 cDNA最大开放读码框所推测的氨基酸序列与已知人、小鼠、大鼠、狗、羊和鸡等真核生物BMP4氨基酸序列进行比较,分别有94.5%、93.1%、91.9%、87.4%、94.2%、79%的同源性。这为克隆其他物种的BMP4基因提供了依据,同时牛骨形态发生蛋白的测序为我们更好的理解牛的生骨机理提供帮助。     

用RACE技术扩增并克隆牛BMP4基因3′端序列

RACE技术是一项扩增基因末端序列的新技术。该研究从牛BMP4基因出发,以牛软骨的RNA为模板,按照不同物种BMP4基因的相似性设计特异引物,运用PCR和RACE技术扩增并获得了特异片段,该片段经PCR、酶切和测序验证,证实所克隆序列为牛BMP4的3′端序列,包含有1170bp组成的开放读码框(ORF),编码389个氨基酸,3′非编码区121bp个核苷酸和poly(A)15。同源性分析结果表明,牛BMP4 cDNA最大开放读码框所推测的氨基酸序列与已知人、小鼠、大鼠、狗、羊和鸡等真核生物BMP4氨基酸序列进行比较,分别有94.5%、93.1%、91.9%、87.4%、94.2%、79%的同源性。这为克隆其他物种的BMP4基因提供了依据,同时牛骨形态发生蛋白的测序为我们更好的理解牛的生骨机理提供帮助。     

Preferential uptake of restriction fragments from a gonococcal cryptic plasmid by competent Neisseria gonorrhoeae

Factors involved in the specificity of DNA uptake by competent Neisseria gonorrhoeae were examined. Host-controlled modification did not affect uptake. Certain restriction fragments of the 4.2 kb gonococcal cryptic plasmid pFA1 and of the replicative form of the bacteriophage M13 were taken up in preference to others, independent of differences in fragment size. A 600 bp fragment from the 4.2 kb plasmid was cloned into pLES2, a gonococcal-Escherichia coli shuttle vector; the 600 bp fragment was taken up into a DNAase-I-resistant state in preference to the vector fragment. A second 370 bp fragment in pFA1 was also taken up preferentially. The 600 bp and 370 bp fragments share a 10 bp sequence, which is found in pFA1 only on fragments that were taken up readily. However, a fragment from M13 which was efficiently taken up did not contain this 10 bp sequence. In addition, this sequence was not sufficient to direct preferential DNA uptake by gonococci, since a recombinant plasmid containing this 10 bp sequence was not taken up appreciably better than the vector plasmid or another recombinant plasmid containing an unrelated 10 bp sequence. Sequence comparisons of the three restriction fragments which were preferentially taken up did not yield any consensus sequences greater than 7 bp. Although it is likely that efficient uptake of DNA by gonococci is determined by DNA structure, a single short sequence could not be found that accounted for specific uptake.     

Cloning,characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

A 3.3 kb HindIII restriction-digest DNA fragment was isolated from a Synechocystis sp. strain PCC6803 subgenomic plasmid library which strongly hybridized to a 349 bp fragment of the icfA (ccaA) gene from Synechococcus sp. strain PCC7942. DNA sequence analysis of the fragment revealed three open reading frames (ORFs), two of which potentially coded for pantothenate synthetase (ORF275) and cytidylate kinase (ORF230). The third, ORF274, was 825 bp in length, encoding a deduced polypeptide of 274 aa (M_{_r}, 30747) that bears 55% sequence identity to the Synechococcus icfA (ccaA) translation product, a -type carbonic anhydrase (CA). A 932 bp EcoRI fragment containing ORF274 was subcloned into an expression vector and the construct was transformed into Escherichia coli for overexpression. Electrometric assays for CA activity revealed that whole cell extracts containing the recombinant protein significantly enhanced the rate of conversion of CO_{_2} to HCO^- _{_3} and that 98% of this catalytic activity was inhibited by ethoxyzolamide, a well-characterized CA inhibitor. Antisera derived against the overexpressed protein recognized a 30.7 kDa protein that was predominantly associated with the isolated carboxysome fraction from Synechocystis. These results provide molecular and physiological evidence for the identification of a ccaA homologue in Synechocystis PCC6803 that encodes a carboxysomal -type CA.     

Molecular markers shared by diverse apomictic Pennisetum species

Two molecular markers, a RAPD (randomly amplified polymorphic DNA) and a RFLP/STS (restriction fragment length polymorphism/sequence-tagged site), previously were found associated with apomictic reproductive behavior in a backcross population produced to transfer apomixis from Pennisetum squamulatum to pearl millet. The occurrence of these molecular markers in a range of 29 accessions of Pennisetum comprising 11 apomictic and 8 sexual species was investigated. Both markers were specific for apomictic species in Pennisetum. The RFLP/STS marker, UGT 197, was found to be associated with all taxa that displayed apomictic reproductive behavior except those in section Brevivalvula. Neither UGT197 nor the cloned RAPD fragment OPC-04₆₀₀ hybridized with any sexually reproducing representatives of the genus. The cloned C04₆₀₀ was associated with 3 of the 11 apomictic species, P. ciliare, P. massaicum, and P. squamulatum. UGT197 was more consistently associated with apomictic reproductive behavior than OPC04₆₀₀ or cloned C04₆₀₀, thus it could be inferred that UGT197 is more closely linked to the gene(s) for apomixis than the cloned C04₆₀₀. The successful use of these probes to survey other Pennisetum species indicates that apomixis is a trait that can be followed across species by using molecular means. This technique of surveying species within a genus will be useful in determining the relative importance of newly isolated markers and may facilitate the identification of the apomixis gene(s).     

A polymerase chain reaction based method for the detection of Culex quinquefasciatus (Diptera: culicidae)

Culex quinquefasciatus Say is the major vector of the filarial parasite Wuchereria bancrofti (Cobbold) which causes lymphatic filariasis in humans. A repetitive DNA sequence from the genome of C. quinquefasciatus has been cloned and completely sequenced. The 693 bp cloned fragment had an A+T content of 72%. Dot matrix analysis of the fragment did not reveal any direct or inverted repeats within it. Southern blot analysis using a variety of restriction enzymes appeared to indicate that the cloned fragment was interspersed within the genome with a copy number of approximately 30,000. A search of the GenBank database did not reveal significant homologies to any previously cloned sequences. Although the probe was sensitive enough to detect picogram quantities of DNA, it was not specific for C. quinquefasciatus, as it hybridized with DNA from other mosquito species, Culex pseudovishnui Colless, Culex gelidus Theobald, Culex tritaeniorhynchus Giles, Anopheles vagus D?nitz and Mansonia uniformis (Theobald). However PCR primers derived from the cloned sequence, IpC, were found to be specific and amplified only C. quinquefasciatus DNA. The optimized PCR assay was found to be very sensitive and was capable of detecting DNA from all stages of C. quinquefasciatus thus making it an ideal diagnostic tool.     

Cloning and sequences of inducible and constitutive macrolide resistance genes in Staphylococcus aureus that correspond to an ABC transporter

A restriction map was made and the DNA sequence was determined for a plasmid, pMC38, derived from the inducible macrolide resistance plasmid pEP2104, that showed constitutive resistance to PMS antibiotics (partial macrolide and streptogramin B antibiotics). A 5. 04 kb SalI-PstI fragment (fragment C) of pMC38, which encoded PMS resistance, was cloned into a shuttle vector, pRIT5, to yield pMR504. The transformant Staphylococcus aureus 4220 (pMR504) exhibited constitutive PMS resistance. Fragment C was subcloned to pUC19 in order to determine the DNA sequence. This sequence was consequently found to contain three open reading frames (ORF1-3), of which ORF3 corresponded to the 63 kDa membrane protein (MsrSA) that expressed PMS resistance. According to DNA sequence comparison of the control region of ORF3 in pMC38 and pEP2104, 44 nucleotides including RBS1 and the leader peptide (MTASMRLK) were deleted on plasmid pMC38. This suggests that the leader peptide is essential for the inducible expression of PMS resistance.     

The replication origin of <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis>

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.     

Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.     

山蛭基因组DNA的提取及其PCR扩增产物的分析

利用PCR技术从海南山蛭体内分离山蛭素（抗凝血蛋白）基因,首先需获得不带有山蛭体表色素且完整的山蛭基因组DNA,本试验通过结合使用SDS-蛋白酶裂解法和CTAB法,有效的去除了山蛭基因组DNA提取过程中难以去除的色素,得到的基因组DNA保持完整,无降解,以之作为模板,进行PCR扩增,获得一个长度约为237bp的特异性片段,此片段与印度山蛭蛭素的cDNA大小几乎一致。初步表明,这个序列没有内含子,而仅是海南山蛭蛭素的编码序列。     

The primary structure and characteristics of ISAfe600, an insertion sequence from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains

A new IS-like element (604 bp) was revealed in the genome of several Acidithiobacillus ferrooxidans strains isolated from diverse biotopes. It includes 26-bp imperfectly matched terminal inverted repeats (TIRs), similar in structure to the TIRs of the ISAfel insertion element. The 60-bp DNA fragment adjacent to the right TIR (TIR_R) exhibits pronounced homology with the similarly located DNA fragments in ISAfel and IST445, as well as with the internal fragment of ISAfel encoding the transposase gene (nucleotides from 254 to 311 bp). The central section of ISAfe600 is unique and exhibits no homology with any prokaryotic DNA. A duplication of 8 bp of the target DNA was found in the ISAfe600 insertion site. One to four copies of ISAfe600 were revealed by Southern hybridization in the genome of A. ferrooxidans strains studied. The number of ISAfe600 copies varies depending on the growth conditions (energy substrate). Since open reading frames big enough to encode transposase are not presert in the structure of ISAfe600, it may be a deficient IS element; its translocation is possibly achieved under control of the ISAfel transposase.     

CAP binding to B and Z forms of DNA.

We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy. The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding. A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer. In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers. A slight preference for the B form was found. These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP.