Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Construction of cDNA libraries for highly efficient DNA sequencing from the 3' end of expressed genes

The majority of expressed sequence tag (EST) sequences available today have been derived from the 5' ends of cDNA clones. Obtaining high-quality DNA sequences from the 3' ends of oligo(dT)-primed cDNA on a large scale has been difficult because of slippage of the DNA polymerase enzyme used in direct PCR and cycle sequencing. With the completion of whole genome sequencing for more and more organisms, mRNA 3'-UTR sequences can be particularly useful for clustering large numbers of ESTs for the effective discrimination of individual genes and gene families. We have identified a flaw in the widely used oligo(dT) primers for cDNA synthesis, and here we describe an improved priming approach to effectively synthesize cDNA devoid of homopolymeric nucleotide stretches from mRNA poly(A) tails to enable highly efficient and reliable DNA sequence determination from 3' mRNA ends. Using this method, we produced a rat lung cDNA library and successfully sequenced the 3' ends of 98% of all attempted clones.     

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.     

Isolation of developmentally regulated genes by differential display screening of cDNA libraries.

mRNA differential display RT-PCR has been extensively used for the isolation of genes differentially expressed between RNA populations. We have assessed its utility for the identification of developmentally regulated genes in plasmid cDNA libraries derived from individual tissues dissected from early mouse embryos. Using plasmid Southern blot hybridisation as a secondary screen, we are able to identify such genes and show by whole-mount in situ hybridisation that their expression pattern is that expected from the differential display profile.     

RT-PCR检测HUTP14A mRNA时排除假基因HUTP14C干扰的方法

人类U3蛋白14C基因(HUTP14C)是人类U3蛋白14A基因(HUTP14A)的假基因。两者转录本序列同源性高达95%。常规RT-qPCR技术在检测HUTP14A mRNA丰度时,HUTP14C的存在会影响检测结果。本研究旨在建立检测HUTP14A mRNA时排除HUTP14C干扰的RT PCR方法。本研究设计出能分别从多种肿瘤细胞DNA和RNA中特异性扩增HUTP14A和HUTP14C的引物,避免假基因HUTP14C对其同源基因HUTP14A检测的干扰。在检测细胞系HUTP14A mRNA时,通过DNaseⅠ消除RNA中污染的HUTP14C DNA,用靶向HUTP14C 3′-UTR的siRNA沉默HUTP14C mRNA后,再用RT PCR检测HUTP14A mRNA丰度,使结果更加准确。在18对肝癌及癌旁组织中,利用特异性引物进行RT PCR检测,HUTP14A和HUTP14C mRNA的表达略高于癌旁组织。本研究提示,针对有假基因存在的功能基因,对其mRNA丰度进行检测时,在提取细胞或组织总RNA后,用DNaseⅠ处理,再用RNA直接进行PCR扩增,排除DNA污染后,再进行RT-PCR或RT-qPCR扩增。大多假基因具有较长的3′-UTR区,在该区域设计siRNA特异性沉默假基因的mRNA后,用RT-qPCR检测功能基因的mRNA丰度,可以排除假基因mRNA的影响。在病理组织中检测功能基因的mRNA丰度时,可以根据假基因和其功能基因的序列差异设计出特异扩增功能基因的引物,从假基因的3′-UTR区设计特异扩增假基因的引物,通过RT-qPCR技术分别检测二者的mRNA。     

Differential regulation of human CYP4A genes by peroxisome proliferators and dexamethasone

HepG2 cells that stably overexpress PPARalpha were used to examine the regulation of the two known human CYP4A genes by Wy14643. Specific PCR amplification across intron 5 and restriction endonuclease analysis indicated that HepG2 cells possess genes corresponding to both the CYP4A11 cDNA and a more recently characterized gene, CYP4A22, that exhibits 95% identity to CYP4A11 in the coding region. These are unlikely to represent alleles because both genes were present in DNA samples from 100 of 100 individuals. Quantitative real-time PCR determined that CYP4A22 mRNA is expressed at significantly lower levels than CYP4A11 mRNA in human liver samples. The PPARalpha agonist Wy14643 induced CYP4A11 mRNA in confluent cultures of HepG2 cells stably expressing the murine PPARalpha-E282G mutant. This mutant exhibits a significantly decreased ligand-independent trans-activation and can be activated by Wy14643 to a level similar to that of wild-type PPARalpha. Dexamethasone induced CYP4A11 mRNA in both control and PPARalpha- E282G-expressing HepG2 cells, indicating that the induction of CYP4A11 by dexamethasone is independent of elevated PPARalpha expression. Wy14643 or dexamethasone induction of CYP4A22 mRNA was not evident in either control or PPARalpha -E282G-expressing HepG2 cells. The results indicate that CYP4A11 expression can be induced by glucocorticoids and peroxisome proliferators.     

Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis

Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific products separated by capillary electrophoresis. A linear relationship between template concentration and fluorescent signal can be demonstrated down to template concentrations in the low aM region, corresponding to approximately 0.04 zmol (24 molecules) per reaction. The technique is shown to be quantitative over five orders of magnitude of template concentration, and average mRNA abundances of approximately 0.01 molecule per cell can be detected. A single predefined set of 320 primers provides 90-95% coverage of all eukaryotic genomes. Analysis of a set of 19 p53-regulated genes in untreated cultures of normal human epithelial cells, derived from three different tissues, revealed a 600-fold range of apparent constitutive expression levels. For most of the genes assayed, good correlations were observed among the expression levels in normal mammary, bronchial, and epidermal epithelial cells.     

Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse.

Fibroblast growth factors (FGFs) can influence the growth and differentiation of cultured cells derived from neuroectoderm, ectoderm or mesenchyme. The FGFs interact with a family of at least four closely related receptor tyrosine kinases that are products of individual genes. To investigate the role of FGFs in the growth and differentiation of embryonic tissues and to determine whether the individual FGF receptor genes might have specific functions, we compared the localization of mRNA for two FGF receptor genes, FGFR1 (the flg gene product) and FGFR2 (the bek gene product), during limb formation and organogenesis in mouse embryos (E9.5-E16.5). Although the two genes were coexpressed in some tissues, the differential expression of FGFR1 and FGFR2 in most embryonic tissues was striking. FGFR1 was expressed diffusely in mesenchyme of limb buds, somites and organ rudiments. In contrast, FGFR2 was expressed predominantly in the epithelial cells of embryonic skin and of developing organs. The differential expression of FGFR1 and FGFR2 in mesenchyme and epithelium respectively, suggests the receptor genes are independently regulated and that they mediate different functions of FGFs during development.     

Amplification and activation of c-Ki-ras oncogene in cell line developed from human pancreas explants.

Amplification and activation of c-Ki-ras gene was studied in normal human pancreas and a cell line (T-3) derived from normal pancreas explants exposed to methylnitrosourea (MNU) for 26 weeks. Normal genomic DNAs from pancreas and derived cell lines showed no transforming activity in NIH 3T3 cells. However, DNAs isolated from tumorigenic cell line derived from MNU treated human pancreas explants transformed NIH 3T3 cells. The hybridization profiles showed that the c-Ki-ras gene was amplified 5 fold in the tumorigenic cells (T-3). The level of mRNA specific to the c-Ki-ras gene was found to be 50-60 fold higher in the malignant cells than in normal human pancreas. These results suggest that higher expression of ras genes is due to gene amplification and/or activation, which is an important step in carcinogenesis.     

Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA

We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.     

Molecular cloning of transcripts that accumulate during the late G1 phase in cultured mouse cells

To identify previously undetected genes that might be involved in later stages of the transition from a quiescent state (G0) to the DNA synthetic phase (S) of murine cells, we set out to isolate cDNA clones derived from mRNAs that appear late in G1 phase in serum-stimulated cells. A lambda-cDNA library was prepared using poly(A)+ RNA from chemically transformed Balb/c 3T3 cells (BP/A31) that had been brought to quiescence and subsequently stimulated for 12 h with serum. From the first screening of approximately 21,000 recombinant phage plaques, about 100 clones were isolated that hybridized to a single-stranded cDNA pool derived from stimulated-cell RNA but not to DNAs made from resting-cell RNA. Eventually, six different clones were identified. The mRNAs from five of these genes increased gradually during the G0 to S transition, in contrast to the "immediate-early" rise of c-myc mRNA or the later rise of thymidine kinase mRNA. These six clones were sequenced and compared to the GenBank database. Clones LG-80, LG-2, and LG-69 are highly homologous to beta-actin, lactate dehydrogenase, and alpha-tubulin. Clones LG-5, LG-61, and LG-74 had no significant homologies to known sequences. A subtractive cDNA library was used to isolate two additional clones, Sub-S1 and Sub-S2; these have homologies to enolase and ribosomal protein L32. Additional studies that examine the function and regulation of these newly identified "late response" genes in the pre-DNA synthesis pathway are in progress.     

Chromosomal Assignments of Novel Genes Expressed in HL60 Granulocytes

By collecting 3'-directed cDNA sequences called gene signatures(GSs) on a large scale, it is possible to make an expressionprofile of genes in a particular tissue, as well as discoveringa number of novel genes. A total of 305 novel GSs collectedfrom granulocytoid cells derived from HL60, a human promyelocyticleukemia cell line, by exposure to dimethyl sulfoxide were radiolabeledand used for Southern blot analyses to determine the copy numberof the corresponding genes. Of these, 198 GSs identified asrepresenting single-copy genes were then used as probes forhybridization analyses using a monochromosomal hybrid cell DNApanel. Sixty-nine of them were assigned to individual chromosomes.These results demonstrate that the chromosomal distributionof the GSs seems not to be proportional to the cytogenetic lengthof each chromosome.