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181.
采用逆转录聚合酶链反应(RT-PCR)从广东省一例慢性丙型肝炎病人血清中获得丙型肝炎病毒(HCV)5'端非编码区(5'NCR)302bp的cDNA片段,经补齐和提纯后插入pUC19质粒,获得的重组体pUN进行序列测定。将pUN的目的基因亚克隆进体外转录载体pSPORTI多克隆位点的EooRI和PstI切点之间,所得重组体pSN线性化后由T_7RNA多聚酶及SP6RNA多聚酶引导体外转录反应,产物经凝胶电泳及特异引物RT-PCR,证实SP6引导的是正义RNA,T7合成的是反义RNA,其大小分别力429bp和362bp。并证实所得RNA力HCV5'NCRcDNA转录而来。获得的HCV5'NCRcDNA和RNA在常规逆转录和PCR步骤中用于设立有效的模板对照,对消除假用性及评估试剂有重要意义。同时,HCV5'NCR体外转录载体的构建可用于制各RNA探针和反义RNA,改进后还可作为定量PCR的竞争性模板。 相似文献
182.
应用离体叶片法,对9个棉花种质进行了鉴定,试验结果表明;种质间抗生性和忌避性差异显著;同株棉花不同部位的叶片对朱砂叶螨的抗生性无显著性差异。通过对叶螨在不同棉花种质上种群增长动态进行系统聚类,可将9个棉花种质划分为3类:斯字棉825-91、杞县86789、鄂棉314、苏联8911为1类,中棉164、潼南接龙棉、新库861517-2、南农NAC90-2为1类,美棉7-15独立为1类。依据朱砂叶螨在不同种质上的种群增长曲线和高峰期螨量增长倍数,可将9个种质划分为3个类型;斯字棉825-91、新库861517-2为抗性类型,潼南接龙棉、美棉7-15、南农NAC90-2为感性类型,其余为中抗类型。从忌避性看:斯字棉825-91、美棉7-15表现出较高的忌避性。 相似文献
183.
E. GHANEM 《Cell biology international》1996,20(10):681-685
Embryonic chick notochords were studied during their metabolically active and involuting periods for the expression of collagen type I and II. The staining was carried out on notochords in vivo at stage 20 and stage 35 and on mesenchyme-contaminated and mesenchyme-free notochords at stage 20, which were cultured in vitro for 6 days. The results show that type II collagen is demonstrable in the notochords, at all the examined stages, both in vivo and in vitro. However, the expression of type I collagen was stage-dependent in vivo and in vitro. At stage 20, the perinotochordal sheath is positively immunostained for collagen type I, but the notochord itself is negative. At stage 35, the perinotochordal sheath as well as the notochord are positively immunostained for collagen type I. The mesenchyme-contaminated and the mesenchyme-free notochords and their sheaths are also positively immunostained for the type I collagen after6 days in vitro. The current results, at late developmental stages, indicate that the involuting notochords express collagen type I, which seems not to be altered by changing the micro-environment in vivo. 相似文献
184.
Joji Sekine Kazuo Sano Masataka Uehara Tsugio Inokuchi 《Biotechnic & histochemistry》1996,71(3):152-156
A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections. 相似文献
185.
用σ ̄(70)或σ ̄(38)和核心酶(E)组成的大肠杆菌RNA聚合酶全酶(Eσ)对四种启动子进行了体外转录。结果表明:lacUV5,rplJ主要被Eσ ̄(70)识别,katE主要被Eσ ̄(38)识别,fic在低盐浓度时被Eσ ̄(70)识别,在高浓度盐时被Eσ ̄(38)识别;Eσ ̄(38)转录特异性启动子所需酶量大于Eσ ̄(70)转录特异性启动子的酶量;在含有分别对σ ̄(70)或σ ̄(38)亲和性大的混和启动子的体外转录中,启动子之间不存在干扰和竞争,转录水平与启动子浓度成正相关。 相似文献
186.
187.
188.
Conversion of a RAPD-generated PCR product,containing a novel dispersed repetitive element,into a fast and robust assay for the presence of rye chromatin in wheat 总被引:19,自引:0,他引:19
H. A. Francis A. R. Leitch R. M. D. Koebner 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1995,90(5):636-642
Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye. 相似文献
189.
W. L. Li P. D. Chen L. L. Qi D. J. Liu 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1995,90(3-4):526-533
A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations. 相似文献
190.
Richard H. Stadler Jovanka Markovic Robert J. Turesky 《Biological trace element research》1995,47(1-3):299-305
The anti- and pro-oxidative effects of phenolic compounds and antioxidants were studied in two different in vitro model systems
utilizing ethyl linoleate and 2′-deoxyguanosine (2′-dG) as oxidative substrates, and a Fenton reaction (H2O2, Fe2+) to initiate oxidation. Oxidation of the biomolecules in both model systems exhibited dose dependency. In the 2′-dG assay,
oxidation was closely related to H2O2 generation, which occurred during autoxidation of the phenolics. Hydroxylating activity was greatly enhanced by Mn2+ and Cu2+, but not by Zn2+ or Co2+. Ethyl linoleate peroxidation was inhibited by low concentrations of catechol, quercitin, and instant coffee. However, peroxidation
was promoted by high concentrations of the same compounds, probably by recycling of chelated inactive Fe3+ to the active Fe2+ state. 相似文献