Semi-quantitative RT-PCR analysis of photoregulated gene expression in marine diatoms

The low cell densities of diatoms and other phytoplankton in culture has precluded the use of classical RNA analysis techniques for routine studies of gene expression in large numbers of samples. This has seriously hampered studies of the basic biology of such organisms. To circumvent this problem, we have developed a high-throughput semi-quantitative RT-PCR-based protocol and used it to monitor expression of a gene encoding a fucoxanthin, chlorophyll a/c-binding protein (FCP) in the centric planktonic diatom Thalassiosira weissflogii. Analysis of FCP gene expression in dark-adapted diatom cultures revealed that mRNA levels increase 5- to 6-fold in response to white light irradiation and peak around 6 to 8 h. To determine the photoreceptors involved in this response action spectra of FCP gene expression were determined using the Okazaki large spectrograph. Responses consistent with the presence of cryptochrome-, rhodopsin- and phytochrome-type receptors could be detected. The apparent presence of phytochrome-mediated responses is of particular interest given the low fluences of red and far-red light wavelengths in the marine environment.     

绵羊BTG1基因的半定量RT-PCR及生物信息学分析

B细胞易位基因1(BTG1基因)是BTG/TOB基因家族的成员之一,在动物细胞的增殖和分化中起重要的作用．利用牛BTG1基因的mRNA序列与绵羊的EST数据库进行Blast检索,并通过序列拼接和逆转录RT-PCR方法首次获得绵羊BTG1基因的部分cDNA序列（GenBank登录号FJ444829）,其片段长度为1 358 bp,包括完整的开放阅读框516 bp,编码171个氨基酸．半定量PCR研究结果表明：BTG1基因在小尾寒羊和陶赛特羊的10种组织中均表达,并具有一致的表达趋势．同源分析结果表明,绵羊BTG1蛋白的氨基酸序列中存在BTG/TOB的保守结构域,并且该蛋白在不同物种间具有很高的保守性．通过生物信息学预测BTG1蛋白功能,发现绵羊BTG1蛋白存在1个跨膜结构域、8个磷酸化位点和1个特异性蛋白激酶磷酸化位点．蛋白质结构同源建模分析表明,绵羊BTG1蛋白具有BTG/TOB蛋白家族的典型空间结构．     

半定量RT-PCR检测运动发酵单胞菌中外源基因转录水平的研究

本研究运用半定量RT-PCR法检测运动发酵单胞菌重组菌中外源基因xylB的转录水平。提取野生型运动发酵单胞菌CP4及其2个重组菌的总RNA, 检测无DNA污染后定量至同一浓度、并反转录为cDNA。观测目的基因xylB和内标基因16S rRNA的PCR扩增曲线、并确定合适的循环数, 选用相同量的cDNA为模板, PCR检测各样本中xylB相对16S rRNA的转录水平。结果表明野生型菌株CP4中xylB基因没有转录, 而两株重组菌中皆有xylB的转录本, 且转录丰度基本一致, 酶活测定也进一步证实该基因在重组菌中有效表达。该方法可用于鉴定运动发酵单胞菌中特定基因的转录水平, 是一种快速有效的检测方法。     

Chromosome-mediated gene transfer of multidrug resistance.

Multidrug resistance can be transferred from drug-resistant LZ Chinese hamster cells to drug-susceptible mouse LTA cells by chromosome-mediated gene transfer. Analysis of genomic DNA demonstrated the transfer of multiple copies of a DNA domain which is amplified in the donor multidrug-resistant cells. The transfer of 10 to 15 copies of the Chinese hamster gene was sufficient to produce a multidrug-resistant phenotype. Chromosome transferents exhibited overexpression of an mRNA of approximately 5 kilobases which has previously been demonstrated to be encoded by the amplified DNA domain of the donor LZ cells. Phenotypic analysis of individual clones selected in adriamycin showed the resistance to be pleiotropic. All clones tested demonstrated similar levels of cross-resistance to the drugs daunorubicin and colchicine. These results indicate that the DNA sequences transferred confer the complete multidrug-resistant phenotype on recipient cells and suggest that multidrug resistance is due to overexpression of the protein encoded by the 5-kilobase mRNA.     

黄瓜RGA基因的半定量RT-PCR表达分析

以黄瓜(Cucumis sativusL.)抗霜霉病品种东农129为材料,利用RT-PCR半定量法研究了接种霜霉病菌(Pseudoperonospora cubensisRostow)、喷施水杨酸(SA)和氯化钙(CaCl2)等不同处理对黄瓜抗病基因类似序列(RGA)表达的影响.结果表明:CsRGA1和CsRGA5基因的表达受霜霉病菌的侵染而启动或加强,外施SA和CaCl2都能够增强其表达;CsRGA4和CsRGA8属于组成型表达基因,其表达可能与霜霉病菌的侵染无关;CsRGA2的表达与外施SA和CaCl2缺乏密切关联.     

Semi-quantitative RT-PCR analysis of environmental influence on P450scc and PNMT mRNA expression in rat adrenal glands.

Environmental influence on brain function, particularly spatial learning and memory, has been extensively investigated, but little is known about the influence of environmental conditions on the functions of peripheral organs. In the present study, the effects of different housing conditions on the steady-state levels of mRNAs encoding cholesterol side-chain cleavage enzyme (cytochrome P450scc) and phenylethanolamine N-methyltransferase (PNMT) in adrenal glands was examined to investigate the environmental influence on both adrenocortical and adrenomedullary functions. Behavioral changes of the animals housed in different conditions were first examined to assess the relevance of environmental manipulation used. In consistent with previous findings, housing of the animals in enriched conditions resulted in the significant reduction of spontaneous motor activity (locomotor activity and rearing) in comparison with housing in isolated conditions, thus indicating the relevance of housing conditions used in this work for investigating the environmental influence on adrenal function. Then, the effects of these housing conditions on P450scc and PNMT mRNA levels in adrenal glands were examined using semi-quantitative RT-PCR method. In comparison with the isolated group, the enriched group showed significantly higher levels of P450scc mRNA. In contrast, PNMT mRNA levels in the enriched group were significantly lower than those in the isolated group. These results propose the possibility that the environmental conditions may cause differential alterations in adrenocortical and adrenomedullary functions, although their possible association with behavioral changes still remains to be elucidated.     

小麦硫转运蛋白基因半定量RT-PCR检测方法的建立

为进一步研究干旱胁迫下小麦硫转运蛋白基因(ST)的表达,选用小麦-αT ubu lin基因(T a)为内参照,提取小麦根系总RNA,反转录cDNA,同批异管对2个基因片段进行PCR扩增.通过对PCR体系中M g2 浓度、退火温度及循环次数的优化和对体系重复性、准确性的分析,最终建立了一个稳定、方便的半定量RT-PCR体系.该体系可用于比较不同小麦样品间硫转运蛋白基因的表达,且因为两对引物均垮内含子,其稳定RT-PCR体系,也为实验室建立小麦mRNA反转录体系提供了参考依据.     

A rapid method to estimate the resistance of cold stored carrot roots to Botrytis cinerea

Inoculating whole carrot roots at 4°C with mycelial/agar discs of the grey mould fungus Botrytis cinerea gives a measure of their resistance and hence storage potential to this pathogen, but results are not obtained for at least 33 days. In the present investigation a more rapid method was used which involved inoculating root slices with spore suspensions containing 5 × 10^3–5 × 10⁶ spores/cm³ at 24°C. Resistance was assessed visually and by a chitin estimation 48 h after inoculation. Both methods were used to measure the resistance of cold stored carrot roots during two storage seasons, 1976/77 and 1977/78. In each season there was a particular inoculum level which most clearly recorded the increasing susceptibility of roots with time in store. In 1976/77 this was 1 × 10⁵ spores/cm³ whereas in 1977/78 it was the lower inoculum concentration of 5 × 10⁴ spores/cm³, suggesting the roots were generally more susceptible in 1977/78 than the previous season. This was in accord with the results from the whole root method of assessment. A chitin estimation proved to be the more sensitive method of assessment for inoculum potential experiments.     

Comparison of mRNA expression levels determined with TaqMan and competitive template RT-PCR

Two methods for measurement of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA expression were compared. Although the relative mRNA levels compared to beta-actin measured with competitive template RT-PCR were different from the data obtained with a TaqMan based PCR, a significant correlation between the two assays was found.     

Isolation and characterization of Drosophila multidrug resistance gene homologs.

Mammalian multidrug-resistant cell lines, selected for resistance to a single cytotoxic agent, display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. These cell lines overproduce a membrane protein, the P-glycoprotein, which is encoded by a member(s) of a multigene family, termed mdr or pgp. The amino acid sequence of the P-glycoprotein predicts an energy-dependent transport protein with homology to a large superfamily of proteins which transport a wide variety of substances. This report describes the isolation and characterization of two Drosophila homologs of the mammalian mdr gene. These homologs, located in chromosomal sections 49EF and 65A, encode proteins that share over 40% amino acid identity to the human and murine mdr P-glycoproteins. Fly strains bearing disruptions in the homolog in section 49EF have been constructed and implicate this gene in conferring colchicine resistance to the organism. This work sets the foundation for the molecular and genetic analysis of mdr homologs in Drosophila melanogaster.