Occludin 1B, a variant of the tight junction protein occludin.
(57/41147)
Occludin and claudin are the major integral membrane components of the mammalian tight junction. Although more than 11 distinct claudins have been identified, only 1 occludin transcript has been reported thus far. Therefore, we searched by reverse transcription-PCR for occludin-related sequences in Madin-Darby canine kidney (MDCK) mRNA and identified a transcript encoding an alternatively spliced form of occludin, designated occludin 1B. The occludin 1B transcript contained a 193-base pair insertion encoding a longer form of occludin with a unique N-terminal sequence of 56 amino acids. Analysis of the MDCK occludin gene revealed an exon containing the 193-base pair sequence between the exons encoding the original N terminus and the distal sequence, suggesting that occludin and occludin 1B arise from alternative splicing of one transcript. To assess the expression and distribution of occludin 1B, an antibody was raised against its unique N-terminal domain. Immunolabeling of occludin 1B in MDCK cells revealed a distribution indistinguishable from that of occludin. Furthermore, occludin 1B staining at cell-to-cell contacts was also found in cultured T84 human colon carcinoma cells and in frozen sections of mouse intestine. Immunoblots of various mouse tissues revealed broad coexpression of occludin 1B with occludin. The wide epithelial distribution and the conservation across species suggests a potentially important role for occludin 1B in the structure and function of the tight junction. (+info)
TOGA: an automated parsing technology for analyzing expression of nearly all genes.
(58/41147)
We have developed an automated, high-throughput, systematic cDNA display method called TOGA, an acronym for total gene expression analysis. TOGA utilizes 8-nt sequences, comprised of a 4-nt restriction endonuclease cleavage site and adjacent 4-nt parsing sequences, and their distances from the 3' ends of mRNA molecules to give each mRNA species in an organism a single identity. The parsing sequences are used as parts of primer-binding sites in 256 PCR-based assays performed robotically on tissue extracts to determine simultaneously the presence and relative concentration of nearly every mRNA in the extracts, regardless of whether the mRNA has been discovered previously. Visualization of the electrophoretically separated fluorescent assay products from different extracts displayed via a Netscape browser-based graphical user interface allows the status of each mRNA to be compared among samples and its identity to be matched with sequences of known mRNAs compiled in databases. (+info)
Cold shock induces the insertion of a cryptic exon in the neurofibromatosis type 1 (NF1) mRNA.
(59/41147)
Alternative splicing is a regulatory process of gene expression based on the flexibility in the selection of splice sites. In this manuscript we present the characterisation of an alternative splicing of the NF1 pre-mRNA induced by cold-shock conditions. We demonstrate that the accuracy of the splicing mechanism was perturbed after keeping samples for a short period of time at room temperature, resulting in the insertion of a 31-bp cryptic exon between exons 4a and 4b of the NF1 mRNA. This alternative splicing is not cell type specific and is not induced by other stress conditions such as heat shock or hyper-osmolarity. The alternative spliced mRNA is efficiently transported to the cytoplasm and it is proven to belong to the poly A(+)mRNA fraction. Previous misleading interpretations about this transcript, together with our finding relating its presence to cold shock and not to the NF1 disease, strongly indicate that this phenomenon should be taken into account in genetic testing when RNA methodology is used for mutation detection. This is the first description of an alternative splicing induced by cold shock in a human pre-mRNA and should provide further insights into the factors that control alternative splicing. (+info)
Identification of human MutY homolog (hMYH) as a repair enzyme for 2-hydroxyadenine in DNA and detection of multiple forms of hMYH located in nuclei and mitochondria.
(60/41147)
An enzyme activity introducing an alkali-labile site at 2-hydroxyadenine (2-OH-A) in double-stranded oligonucleotides was detected in nuclear extracts of Jurkat cells. This activity co-eluted with activities toward adenine paired with guanine and 8-oxo-7,8-dihydroguanine (8-oxoG) as a single peak corresponding to a 55 kDa molecular mass on gel filtration chromatography. Further co-purification was then done. Western blotting revealed that these activities also co-purified with a 52 kDa polypeptide which reacted with antibodies against human MYH (anti-hMYH). Recombinant hMYH has essentially similar activities to the partially purified enzyme. Thus, hMYH is likely to possess both adenine and 2-OH-A DNA glycosylase activities. In nuclear extracts from Jurkat cells, a 52 kDa polypeptide was detected with a small amount of 53 kDa polypeptide, while in mitochondrial extracts a 57 kDa polypeptide was detected using anti-hMYH. With amplification of the 5'-regions of the hMYH cDNA, 10 forms of hMYH transcripts were identified and subgrouped into three types, each with a unique 5' sequence. These hMYH transcripts are likely to encode multiple authentic hMYH polypeptides including the 52, 53 and 57 kDa polypeptides detected in Jurkat cells. (+info)
Cloning and characterisation of the Sry-related transcription factor gene Sox8.
(61/41147)
SOX proteins form a large family of transcription factors related by a DNA-binding domain known as the HMG box. Some 30 Sox genes have been identified in mammals and orthologues have been found in a wide range of other metazoans. Sox genes are highly conserved and are known to play important roles in embryonic development, including roles in gonadal, central nervous system, neural crest and skeletal development. Several SOX genes have been implicated in human congenital diseases. We report here the isolation of Sox8 and its characterisation in mice and humans. This gene has a remarkably similar primary structure and genomic organisation to the campomelic dysplasia gene SOX9 and the Waardenburg-Shah syndrome gene SOX10. SOX8 protein is able to bind to canonical SOX target DNA sequences and activate transcription in vitro through two separate trans -activation regions. Further, Sox8 is expressed in the central nervous system, limbs, kidneys, gonads and craniofacial structures during mouse embryo development. Sox8 maps to the t complex on mouse chromosome 17 and to human chromosome 16p13.3, a region associated with the microphthalmia-cataract syndrome CATM and the alpha-thalassemia/mental retardation syndrome ATR-16. (+info)
Analysis of the yeast transcriptome with structural and functional categories: characterizing highly expressed proteins.
(62/41147)
We analyzed 10 genome expression data sets by large-scale cross-referencing against broad structural and functional categories. The data sets, generated by different techniques (e.g. SAGE and gene chips), provide various representations of the yeast transcriptome (the set of all yeast genes, weighted by transcript abundance). Our analysis enabled us to determine features more prevalent in the transcriptome than the genome: i.e. those that are common to highly expressed proteins. Starting with simplest categories, we find that, relative to the genome, the transcriptome is enriched in Ala and Gly and depleted in Asn and very long proteins. We find, furthermore, that protein length and maximum expression level have a roughly inverse relationship. To relate expression level and protein structure, we assigned transmembrane helices and known folds (using PSI-blast) to each protein in the genome; this allowed us to determine that the transcriptome is enriched in mixed alpha-beta structures and depleted in membrane proteins relative to the genome. In particular, some enzymatic folds, such as the TIM barrel and the G3P dehydrogenase fold, are much more prevalent in the transcriptome than the genome, whereas others, such as the protein-kinase and leucine-zipper folds, are depleted. The TIM barrel, in fact, is overwhelmingly the 'top fold' in the transcriptome, while it only ranks fifth in the genome. The most highly enriched functional categories in the transcriptome (based on the MIPS system) are energy production and protein synthesis, while categories such as transcription, transport and signaling are depleted. Furthermore, for a given functional category, transcriptome enrichment varies quite substantially between the different expression data sets, with a variation an order of magnitude larger than for the other categories cross-referenced (e.g. amino acids). One can readily see how the enrichment and depletion of the various functional categories relates directly to that of particular folds. (+info)
A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells.
(63/41147)
We describe a rapid cell-based genetic screen using fission yeast for identifying efficient gene suppression constructs (GSCs) from large libraries (10(5)) for any target sequence for use in human cells. In this system, target sequences are fused to the 5' end of the lacZ reporter gene and expressed in yeast. Random fragment expression libraries derived from the target sequence are screened in the fusion gene-expressing strain using the lacZ gene-encoded colony color phenotype. We demonstrate the utility of this screening assay by identifying a range of different GSCs for the fission yeast ura4 gene and human c-myc and Chk1 sequences, including rare efficient suppressors. GSCs specific for c-myc were shown to regulate expression of both a c-myc-lacZ fusion gene and the endogenous c-myc gene in human cells. (+info)
Genome changes and gene expression in human solid tumors.
(64/41147)
Genome-wide analysis techniques such as chromosome painting, comparative genomic hybridization, representational difference analysis, restriction landmark genome scanning and high-throughput analysis of LOH are now accelerating high-resolution genome aberration localization in human tumors. These techniques are complemented by procedures for detection of differentially expressed genes such as differential display, nucleic acid subtraction, serial analysis of gene expression and expression microarray analysis. These efforts are enabled by work from the human genome program in physical map development, cDNA library production/sequencing and in genome sequencing. This review covers several commonly used large-scale genome and gene expression analysis techniques, outlines genomic approaches to gene discovery and summarizes information that has come from large-scale analyses of human solid tumors. (+info)