mRNA差异显示条件的优化

运用优化的mRNA差异显示技术分离受内生真菌诱导的差异基因。优化差异显示条件表现在增如指定引物和随机引物的长度、改变PCR参数和再扩增程序、运用银染显色等。应用这些条件共获得7个阳性差异片段。用未优化的PCR程序1筛选35条差异带,得到3个两端均为随机引物的差示片段。而用优化的PCR程序2,52条差异带中得到9条只能用锚定引物和随机引物才能扩增出的片段。地高辛标记的反向-Northern鉴定为阳性后进行克隆和测序。PCR方法1所得的3个差示片段均无开放的阅读框。PCR程序2得到7个差异表达的基因中,2个为已知基因,5个为未知基因。因此可运用优化的差显技术分离差异表达的基因。     

A new assay for proteases using fluorescent labeling of proteins

A simple assay for proteases based on the fluorescent labeling of insoluble proteins (fibrin) or of soluble casein by 2-methoxy-2,4-diphenyl-3(2H)furanone has been developed. Fluorescence of the liberated peptide-fluorophors resulting from enzymatic hydrolysis is easily measured in the supernatant after separation of the unreacted fluorescent fibrin by centrifugation or from unreacted casein-fluorophor by acid precipitation. Nanogram quantities of trypsin, chymotrypsin, and elastase can be measured.     

A statistical model providing comprehensive predictions for the mRNA differential display

MOTIVATION: Differential display (DD) or arbitrarily primed fingerprinting serves to identify differentially expressed genes, but these techniques cannot determine how many of the theoretically available genes have been uncovered. Previous mathematical models are unsatisfying as they are not suitable to analyze experimental data. RESULTS: In the present study, we provide a statistical model based on the redundancy of cDNA fragments amplified during DD experiments. This model is applicable to any DD and predicts (1) the total number of genes expressed in a sample cell type or tissue, (2) the number of differentially expressed genes, (3) the coverage obtained with any given number of primer combinations. In a DD experiment comparing two developmental stages of the post natal rat inner ear, we estimated the total number of differentially expressed genes accessible by DD to be 445, and the number of primer combinations required to uncover 90% of these to be 127. AVAILABILITY: The algorithms were implemented in Matlab (The Mathworks, Inc., Natick, MA) environment and are available at www.physiologie.uni-freiburg.de/download.html CONTACT: ellen.reisinger@physiologie.uni-freiburg.de.     

A rapid protocol for the authentication of isolated differential display RT-PCR CDNAs.

The most time-consuming and problematic step in the overall DDRT-PCR technique is the confirmation that the isolated cDNA clone represents a differentially expressed gene. We have previously suggested that the majority of apparent false positives generated by DDRT-PCR do in fact result from the PCR reamplification of cDNA species which co-migrate with the cDNA of interest, and we have outlined a procedure to effectively eliminate these from further study. However, in situations where RNA is limiting, it is still desirable to confirm that a purified cDNA amplicon does, in fact, represent the originally observed differentially expressed gene prior to embarking on expression studies.     

Robust ordered mRNA differential display: an improved method for global gene expression profiling

Global gene expression profiling (GGEP) plays a pivotal role in biological research. We developed an improved GGEP method called "robust ordered mRNA differential display (RoDD)" by combining mRNA differential display (DD) and complementary DNA amplified fragment length polymorphisms (cDNA-AFLP) using elaborately designed primers and a poly (dT:A) replacement technique. Redundancy was minimized by bead-based isolation and coverage was improved by using restriction enzymes that recognized 4-bp sites. This method offers the common virtues of gel-based methods along with the reliability of cDNA-AFLP. The most significant advantage of RoDD over current gel-based methods is greatly improved coverage and minimized redundancy.     

Improved method for differential expression proteomics using trypsin-catalyzed 18O labeling with a correction for labeling efficiency

Quantitative strategies relying on stable isotope labeling and isotope dilution mass spectrometry have proven to be a very robust alternative to the well established gel-based techniques for the study of the dynamic proteome. Postdigestion 18O labeling is becoming very popular mainly due to the simplicity of the enzyme-catalyzed exchange reaction, the peptide handling and storage procedures, and the flexibility and versatility introduced by decoupling protein digestion from peptide labeling. Despite recent progresses, peptide quantification by postdigestion 18O labeling still involves several computational problems. In this work we analyzed the behavior of large collections of peptides when they were subjected to postdigestion labeling and concluded that this process can be explained by a universal kinetic model. On the basis of this observation, we developed an advanced quantification algorithm for this kind of labeling. Our method fits the entire isotopic envelope to parameters related with the kinetic exchange model, allowing at the same time an accurate calculation of the relative proportion of peptides in the original samples and of the specific labeling efficiency of each one of the peptides. We demonstrated that the new method eliminates artifacts produced by incomplete oxygen exchange in subsets of peptides that have a relatively low labeling efficiency and that may be considered indicative of false protein ratio deviations. Finally using a rigorous statistical analysis based on the calculation of error rates associated with false expression changes, we showed the validity of the method in the practice by detecting significant expression changes, produced by the activation of a model preparation of T cells, with only 5 microg of protein in three proteins among a pool of more than 100. By allowing a full control over potential artifacts, our method may improve automation of the procedures for relative protein quantification using this labeling strategy.     

Linear dynamic range for signal detection in fluorescent differential display

We have determined the linear dynamic range in signal detection by Fluorescent Differential Display (FDD) using conditionally induced mRNA expression of the p53 tumor-suppressor gene as a control. By serial spiking of p53-induced RNA into that of non-induced RNA, we were able to quantitatively measure up to 100-fold change in p53 mRNA expression level. The linear dynamic range of signal detection per mRNA message was determined to be from 1000 up to 20,000 in fluorescence signal, in which the signals for the majority of mRNAs reside. Thus, FDD can be used to accurately quantify differences in mRNA expression among eukaryotic cells.     

Heterogeneous expression of mRNA in rat liver lobules as detected by differential display

Although liver hepatocytes appear to be uniform histologically, they are considerably heterogeneous with respect to their individual physiological capacities. In order to find still unknown genes that are heterogeneously expressed and with the aim of evaluating the usefulness of the differential display technique for this purpose, we performed differential displays with mRNA isolated from hepatocytes from the periportal and pericentral zone of the rat liver. In this way we identified at least two mRNAs exclusively expressed in the pericentral fraction. Sequence analysis revealed that the corresponding genes encode proteins with proline-glutamate dipeptide repeats similar to ones previously identified in rat pheochromocytoma and brain. In situ hybridization confirmed the heterogeneous distribution of the mRNA. Only one to two cell lines surrounding the terminal hepatic venules were positive, strongly resembling the heterogeneous expression of the enzyme glutamine synthetase. Our work demonstrates that the differential display method is a useful tool for the identification of genes that are differentially expressed in individual parenchymal cells. In fact, our results prove that differential display technology can be used for the identification of cellular markers for distinct subpopulations of cells in a given tissue.     

Identification of a gibberellin-induced gene in deepwater rice using differential display of mRNA

Differential display of mRNA was employed to identify gibberellin (GA)-regulated genes in deepwater rice. One of the first differentially displayed products identified was shown to be ten-fold induced after start of GA treatment. The sequence of the clone shows complete amino acid identity with histone H3, and its increased mRNA level correlates with the onset of DNA synthesis. We also identified a gene whose expression pattern did not change over the course of treatment with GA and can be used as standard to correct for loading differences on northern blots.     

Changes in mRNA expression in mouse postnatal cochlea by differential display method.

We compared mRNA expression by mRNA differential display method in postnatal day 11 (P11), P13 and adult C3H/HeJ mouse cochlea. Forty-seven bands were differentially displayed on polyacrylamide gel when 27 patterns of PCR primer sets were used, and 24 of them showed a remarkable increase within only two days (P11 and P13). DNA sequences of the bands were analyzed for homology to known genes using the BLAST search. Most of the clones were identical to sequences of functions unknown. However, one of the clones showing an increase of mRNA expression between P11 and P13 was identified as mouse TIS7 which is known to markedly increase during differentiation of PC-12 cells to neurons by NGF-treatment. TIS7 expression may be involved in differentiation of neuronal progenitor cells to spiral ganglion cells by the initial sound input. The comparison among P11, P13 and adult mouse cochlear mRNA expressions investigated the genes involved in the various growing stages of the postnatal cochlea.     

Analysis of a mod B mutant in Dictyostelium discoideum using mRNA differential display

Three differential cDNAs of Dictyostelium, not detected in the mod B mutant defective in O-glycosylation, were isolated by using an mRNA differential display. These cDNAs encode a protein tyrosine kinase, an adenylyl cyclase and a putative protein kinase C inhibitor whose expression is developmentally regulated.     

Screening for ribosomal-based false positives following prokaryotic mRNA differential display

Differential display (DD) and the closely related RNA arbitrarily primed PCR (RAP-PCR) have become the molecular tools of choice for identifying and isolating differentially expressed genes in both eukaryotic and prokaryotic systems. However, one of the current drawbacks of both techniques is the high number of false positives generated. In prokaryotic applications, the many false positive typically generated by DD are subsequently identified as rRNAs because of their greater abundance compared to mRNAs. To circumvent this problem, full-length 16S and 23S rDNA probes, derived from Pseudomonas putida G7 and Pseudomonas aeruginosa FRD1, respectively, were used as a prescreening approach to discriminate between those bands, which appear to be differentially expressed mRNAs, but in fact are rRNAs, following prokaryotic mRNA DD.     

Cytokinin-induced gene expression in cultured green cells of Nicotiana tabacum identified by fluorescent differential display

The cell growth and plastid development of cultured green tobacco cells were maintained by the phytohormone cytokinin. After subculture into cytokinin-free medium, when cytokinin treatment was resumed, physiological changes induced by cytokinin were analyzed. Changes in chlorophyll biosynthesis and photosynthetic gene expression were observed 1 week after cytokinin induction, and changes in cell growth were observed 2 weeks after cytokinin induction. Two cytokinin-induced genes (cig) were isolated from these cells using the fluorescent differential display technique. Northern analysis confirmed that expression of these cig was induced by both natural and synthetic cytokinins. The expression of cig1 was also induced by abscisic acid, and its cDNA sequence was similar to the proline dehydrogenase gene. The expression of cig2 is specific to cytokinin and is not induced by other phytohormones. The amino acid sequence encoded by cig2 is similar to the GDP/GTP exchange factor eIF2B, which regulates translation initiation. The expression of these cig suggests a complex induction system involving cytokinin and other phytohormones.