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cDNA Probes (cdna + probe)
Selected AbstractsImmune activation upregulates lysozyme gene expression in Aedesaegypti mosquito cell cultureINSECT MOLECULAR BIOLOGY, Issue 6 2000Y. Gao Abstract After stimulation with heat-killed bacteria, cultured cells from the mosquito Aedesaegypti (Aag-2 cells) secreted an induced protein with a mass of , 16 kDa that cross-reacted with antibody to chicken egg lysozyme. To investigate whether lysozyme messenger RNA is induced in bacteria-treated cells, we used polymerase chain reaction-based approaches to obtain the complete lysozyme cDNA from Aag-2 cells. The deduced protein contained 148 amino acids, including a 23 amino acid signal sequence. The calculated mass of the precursor protein is 16 965 Da, which is processed to yield a mature lysozyme of 14 471 Da with a calculated pI of 10.1. The lysozyme from Ae. aegypti shared 50% amino acid identity with lysozymes from Anophelesgambiae and Anophelesdarlingi, which in turn shared 70% identity between each other. Northern analysis with the lysozyme cDNA probe showed induction of a 1.3 kb messenger RNA during the first 3 h after treatment of Aag-2 cells with heat-killed bacteria, followed by maximal expression 12,36 h after treatment. Southern analysis suggested that the gene likely occurs as a single copy in the genome of Aag-2 cells. [source] Characterization of genomic DNA encoding cecropins from an Aedes albopictus mosquito cell lineINSECT MOLECULAR BIOLOGY, Issue 1 2002D. Sun Abstract We used cDNA probes from Aedes albopictus mosquito cecropins AalCecA, B, and C to obtain genomic DNA copies and flanking DNA. Two gene copies (AalCecA1 and A2, AalCecB1 and B2, AalCecC1 and C2) encoding each of the three mature cecropin peptides were recovered. All these genes had a similar organization, into two exons interrupted by a single short intron. AalCecA1 and AalCecA2 encode mature protein products that differ by one amino acid residue, while AalCecB1 and AalCecB2, AalCecC1 and AalCecC2 encode identical mature cecropin peptides, respectively. The AalCecB and C gene pairs each share a common intergenic region of approximately 1 kb, with the two coding regions transcribed in opposite directions. With the exception of small insertions/deletions, the intergenic spacer region was highly conserved between the B1/C1 and B2/C2 clones. In transfected cells, 0.8 kb of upstream sequence was sufficient for inducible expression of AalCecA1. Within this region, a 28 bp sequence at positions ,192 to ,165 upstream of the transcription initiation site was found to contain a potential regulatory element. In electrophoretic mobility shift assays, synthetic double-stranded DNA containing this 28 bp sequence retarded protein in cytoplasmic and nuclear extracts from C7-10 cells. [source] Heparin and Heparan Sulfate BiosynthesisIUBMB LIFE, Issue 4 2002Kazuyuki Sugahara Abstract Heparan sulfate is one of the most informationally rich biopolymers in Nature. Its simple sugar backbone is variously modified to different degrees depending on the cellular conditions. Thus, it matures to have an enormously complicated structure, which most likely exhibits a considerable number of unique overlapping sequences with peculiar sulfation profiles. Such sequences are recognized by specific complementary proteins, which form a huge group of "heparin-binding proteins," and the sugar sequences in turn support unique functions of the respective proteins through specific interactions. The heparan sulfate sequences are not directly encoded by genes, but are created by elaborate biosynthetic mechanisms, which ensure the generation of these indispensable sequences. In heparan sulfate biosynthesis, the tetrasaccharide sequence (GlcA-Gal-Gal-Xyl-), designated the protein linkage region, is first assembled on a specific Ser residue at the glycosaminoglycan attachment site of a core protein. A heparan sulfate chain is then polymerized on this fragment by alternate additions of GlcNAc and GlcA through the actions of glycosyltransferases with overlapping specificities encoded by the tumor suppressor EXT family genes. Then follow various modifications by N -deacetylation and N -sulfation of glucosamine, C5-epimerization of GlcA and multiple O -sulfations of the component sugars. Recent studies have achieved purification of several, and molecular cloning of most, of the enzymes responsible for these reactions. Some of these enzymes are bifunctional. The availability of cDNA probes has facilitated elucidation of the crystal structures for two of the biosynthetic enzymes, demonstration of their intracellular location, and their occurrence in complexes to achieve rapid and efficient synthesis of complex sugar sequences. Genomic structure and transcript analysis have shown the existence of multiple isoforms for most of the sulfotransferases. Many aspects of the heparan sulfate biosynthetic scheme are shared by the structural analog heparin, which is synthesized in mast cells and some other mammalian cells and is several-fold higher degree of polymerization and more extensive modification than heparan sulfate. [source] Genomic repertoire of human mesangial cells: comprehensive analysis of gene expression by cDNA array hybridizationNEPHROLOGY, Issue 4 2000Naohiro Yano SUMMARY: Knowing when and where a gene is expressed in a cell often provides a strong clue as to its physiological role. It is estimated the human genome contains 80 000,100 000 genes. Assessment of gene activity on a global genome-wide scale is a fundamental and newly developed experimental strategy to expand the scope of biological investigation from a single gene to studying all genes at once in a systematic way. Capitalizing on the recently developed methodology of cDNA array hybridization, we monitored the simultaneous expression of thousands of genes in primary human mesangial cells. Complex ,- 33P-labelled cDNA probes were prepared from cultured mesangial cells. The probe was hybridized to a high-density array of 18 326 paired target genes. The radioactive hybridization signals were analysed by phosphorimager. Bioinformatics from public genomic databases was utilized to assign a chromosomal location of each expressed transcript. Approximately 7460 different gene transcripts were detected in mesangial cells. Close to 13% (957 genes) were full-length mRNA human transcripts (HTs), the remainder 6503 being expressed sequence tags (ESTs). Using special imaging computer software, the transcriptional level of the 957 HTs was compared with the expression of the ribosomal protein S28 (housekeeping gene). The HTs were also classified by function of the gene product and listed with information on their chromosomal loci. To allow comparison between clinical and experimental studies of gene expression, the detected human gene transcripts were cross-referenced to orthologous mouse genes. Thus, the presented data constitute a quantitative preliminary blueprint of the transcriptional map of the human mesangial cell. The information may serve as a resource for speeding up the discovery of genes underlying human glomerular diseases. The complete listing of the full-length expressed genes is available upon request via E-mail: (Abdalla_Rifai@Brown.edu). [source] Molecular fingerprinting of TGFß-treated embryonic maxillary mesenchymal cellsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 4 2003M.M. Pisano Abstract The transforming growth factor-ß (TGFß) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGFß on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGFß in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGFß action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's AtlasTM Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGFß-treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane-based cDNA array (1178 genes) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either TGFß-treated or vehicle-treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImageTM analysis in order to determine differences in gene expression between control and TGFß-treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up-regulated, while those of 8 other genes were down-regulated, by a factor of twofold or greater in response to TGFß. Affected genes could be grouped into three general functional categories: transcription factors and general DNA-binding proteins; growth factors/signaling molecules; and extracellular matrix and related proteins. The extent of hybridization of each gene was evaluated by comparison with the abundant, constitutively expressed mRNAs: ubiquitin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ornithine decarboxylase (ODC), cytoplasmic beta-actin and 40S ribosomal protein. No detectable changes were observed in the expression levels of these genes in response to TGFß treatment. Gene expression profiling results were verified by Real-Time quantitative polymerase chain reaction. Utilization of cDNA microarray technology has enabled us to delineate a preliminary transcriptional map of TGFß responsiveness in embryonic maxillary mesenchymal cells. The profile of differentially expressed genes offers revealing insights into potential molecular regulatory mechanisms employed by TGFß in orchestrating craniofacial ontogeny. [source] Transcriptionally active transposable elements in recent hybrid sugarcaneTHE PLANT JOURNAL, Issue 5 2005Paula G. de Araujo Summary Transposable elements (TEs) are considered to be important components of the maintenance and diversification of genomes. The recent increase in genome sequence data has created an opportunity to evaluate the impact of these active mobile elements on the evolution of plant genomes. Analysis of the sugarcane transcriptome identified 267 clones with significant similarity to previously described plant TEs. After full cDNA sequencing, 68 sugarcane TE clones were assigned to 11 families according to their best sequence alignment against a fully characterized element. Expression was further investigated through a combined study utilizing electronic Northerns, macroarray, transient and stable sugarcane transformation. Newly synthesized cDNA probes from flower, leaf roll, apical meristem and callus tissues confirm previous results. Callus was identified as the tissue with the highest number of TEs being expressed, revealing that tissue culture drastically induced the expression of different elements. No tissue-specific family was identified. Different representatives within a TE family displayed differential expression patterns, showing that each family presented expression in almost every tissue. Transformation experiments demonstrated that most Hopscotch clone-derived U3 regions are, indeed, active promoters, although under a strong transcriptional regulation. This is a large-scale study about the expression pattern of TEs and indicates that mobile genetic elements are transcriptionally active in the highly polyploid and complex sugarcane genome. [source] Differential expression of the GTL2 gene within the callipyge region of ovine chromosome 18ANIMAL GENETICS, Issue 5 2001C. A. Bidwell The inheritance pattern of the skeletal muscle hypertrophy phenotype caused by the callipyge gene has been characterized as polar overdominance. We hypothesized that this trait may be caused by a gain or loss of gene expression because of the reversible nature of the phenotype in paternal vs. maternal inheritance. Suppression subtraction cDNA probes were made from skeletal muscle mRNA of normal (NN) and callipyge (CPatNMat) animals and hybridized to Southern blots containing bacterial artificial chromosomes (BACs) that comprise a physical contig of the callipyge region. The CN,NN probes hybridized to two ovine and seven bovine BACs. Sequence analysis of fragments within those BACs indicated short regions of similarity to mouse gene trap locus (gtl2). Northern blots analysis of RNA from hypertrophy-responsive muscles show a population of GTL2 mRNA centred around 2.4 kb that were abundantly expressed in 14-day prenatal NN and CPatNMat lambs but were down-regulated in day 14 and day 56 postnatal NN lambs. The expression of GTL2 remained elevated in 14- and 56-day-old CPatNMat lambs as well as in 56-day-old NPatCMat and CC lambs. Expression of GTL2 in the supraspinatus, which does not undergo hypertrophy, was very low for all genotypes and ages. Isolation of cDNA sequences show extensive alternative splicing and a lack of codon bias suggesting that GTL2 does not encode a protein. The mutation of the callipyge allele has altered postnatal expression of GTL2 in muscles that undergo hypertrophy and will help identify mechanisms involved in growth, genomic imprinting and polar overdominance. [source] | |||||||||||||