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Expression analysis of SIX3 and SIX6 in human tissues reveals differences in expression and a novel correlation between the expression of SIX3 and the genes encoding isocitrate dehyhrogenase and cadherin 18.

SIX3 and SIX6 are transcription factors expressed during early stages of eye development. Limited expression data for SIX3 and SIX6 are available in the literature but, to date, there are no reports of the relative levels of expression of these genes throughout the human body and in adult tissues in particular. In this paper, we report extensive real-time quantitative PCR analyses of SIX3 and SIX6 expression in many different tissues of the adult human body, including ocular tissues, and a comparison of expression data with that of many other genes to identify similarity in expression. Using this powerful technique, we have detected a novel statistical correlation between the spatial distribution and the quantitative expression of SIX3 and 5 other transcripts including IDH1, the gene encoding the NADP(+)-dependent enzyme isocitrate dehydrogenase, and cadherin 18, type 2 (CDH14). Our data demonstrate that this novel technique can be used to generate hypotheses by comparison of gene expression profiles to identify possible interactions between genes or gene products.

Adult↗

cDNA cloning, genomic structure, chromosomal mapping and expression analysis of ADIPOQ (adiponectin) in chicken.

We report sequence, tissue expression and map position data for the adiponectin gene (ADIPOQ) in chicken. This gene is involved in adipocyte differentiation in mammals and may play similar roles in chicken. The complete coding sequence of chicken ADIPOQ encodes a 244 amino acid protein. The coding region of the chicken ADIPOQ is organized in two exons and spans an approximately 2.1 kb genomic region. Domain/motif structures of the adiponectin protein among chicken and mammalian homologs are highly conserved. Reverse transcription-PCR revealed chicken ADIPOQ mRNA displayed high expression levels in fat tissue, heart, stomach and skin and weak expression in muscle. Analysis of a radiation hybrid panel indicated that ADIPOQ showed the most significant linkage to FOXL2 (LOD = 12.63, 12 cR) and ATSV (LOD = 7.53, 3 cR) both of which are located on chicken chromosome 9. Information provided in this study will be useful in further studies to determine the role the adiponectin gene plays in the regulation of glucose and lipid metabolism and body weight in chicken.

Adiponectin↗

A cDNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in Helicoverpa armigera: molecular characterization and expression analysis associated with pupal diapause.

The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9-10 kDa highly conserved multifunctional protein that plays important roles in GABA(A) receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera.

Amino Acid Sequence↗

Gene expression analysis to identify mRNA markers of cardiac myxoma.

cDNA expression arrays were used to identify mRNA expression markers for cardiac myxoma. The RNA profile analysis suggests that cardiac myxoma should be considered as a stand-alone tissue rather than a pathological modification of particular normal tissue. The analysis reveals a set of genes which are highly and steadily expressed in cardiac myxomas and can serve as an mRNA expression markers of the tumour. Marker status of selected genes was confirmed by reverse transcriptase polymerase chain reaction analysis. Genes MIA (melanoma inhibitory activity) and PLA2G2A (phospholipase A2, group IIA) show the highest specificity as cardiac myxoma markers, since they have more than 10-fold higher RNA level in cardiac myxomas than in any one of 15 normal tissues tested. Among markers of myxoma at least three are participants of phospholipid metabolism: ANXA3, PLA2G2A, and phospholipid transfer protein. Tissue inhibitor of metalloproteinase 1 and secretory leucocyte protease inhibitor are inhibitors of proteases degrading extracellular matrix proteins and participating in cell proliferation regulation. MIA, SPP1, fibromodulin are modulators or participants of the interaction between extracellular matrix proteins and their cell surface receptors. SOX9 is a transcription factor required for chondrocyte differentiation. Calretenin (CALB2) is an intracellular calcium-binding protein with poorly understood function.

Adolescent↗

Molecular cloning and expression analysis of rat Rgs12 and Rgs14.

We report the cloning of two novel rat regulators of G-protein signaling (RGS) cDNAs using a degenerate PCR strategy. The rRgs12 and rRgs14 cDNAs encode predicted polypeptides of 1387 and 544 amino acids, respectively. We have also identified the human orthologue of rRgs12 by alignment of cosmid sequences in the database which map the human RGS12 gene to chromosome 4p16.3. Furthermore, we identified human ESTs with high homology to rRgs14 which map to human chromosome 5qter. Northern blot analysis indicates that rRgs14 is expressed at high levels in brain, lung, and spleen, whereas rRgs12 is expressed at high levels in brain and lung and lower levels in testis, heart, and spleen. Analysis of the predicted rRGS12 and rRGS14 polypeptides indicates that they are closely related and possess regions of homology outside of the conserved RGS domain. We have also identified conserved regions in RGS12 which are similar to protein domains found in mouse rhophilin and coiled-coil proteins suggesting possible interactions with ras-like G-proteins.

Amino Acid Sequence↗

Identification of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis.

To identify genes that are differentially over-expressed in Small Cell Lung Carcinoma (SCLC) we have used a combination of suppression subtractive hybridization and cDNA microarray to analyse the expression profiles of 2400 cDNAs clones. Genes that are over-expressed in SCLC were identified using 32 pairs of fluorescence-labeled cDNA samples representing various lung tumors and normal tissues. This comprehensive approach has resulted in the identification of 209 genes that are differentially over-expressed in SCLC. Quantitative real-time PCR was used to further validate the expression of 43 genes in SCLC tumors and various normal tissues. Discussed in this report are nine genes, which showed the most promising SCLC tumor to normal tissue differential expression profiles, including seven known and two novel genes. The large number of differentially expressed genes identified from this analysis and the characterization of these genes will provide valuable information in better understanding the biology of SCLC and help us in developing these gene products as potential targets for diagnostic as well as therapeutic usage.

Carcinoma, Small Cell↗

Behavior of osteoblast, adipocyte, and myoblast markers in genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro.

Several genes, such as alkaline phosphatase, osteocalcin, and Cbfa1/Osf2, are known to be regulated during osteoblastic differentiation and are commonly used as "osteoblast markers" for in vitro or in vivo studies. The number of these genes is very limited, however, and it is of major interest to identify new genes that are activated or repressed during the process of osteoblast differentiation and bone formation as well as to extend the available information on gene families relevant to this particular differentiation pathway. To identify such genes, we have implemented a genome-wide analysis by determining changes in expression levels of 27,000 genes during in vitro differentiation of primary osteoblasts isolated from mouse calvaria. This study focuses on the description of the analytical and filtering process applied; on the transcriptional analysis of well-established "bone," "adipocyte," and "muscle" pathway markers; and on a description of the regulation profiles for genes recently described in the Skeletal Gene Database. We also demonstrate that new array technologies constitute reliable and powerful tools to monitor the transcription of genes involved in osteoblastic differentiation, allowing a more integrated vision of the biological pathways regulated during osteoblast commitment, differentiation, and function.

Adipocytes↗

Gene expression analysis with the parametric bootstrap.

Recent developments in microarray technology make it possible to capture the gene expression profiles for thousands of genes at once. With this data researchers are tackling problems ranging from the identification of 'cancer genes' to the formidable task of adding functional annotations to our rapidly growing gene databases. Specific research questions suggest patterns of gene expression that are interesting and informative: for instance, genes with large variance or groups of genes that are highly correlated. Cluster analysis and related techniques are proving to be very useful. However, such exploratory methods alone do not provide the opportunity to engage in statistical inference. Given the high dimensionality (thousands of genes) and small sample sizes (often <30) encountered in these datasets, an honest assessment of sampling variability is crucial and can prevent the over-interpretation of spurious results. We describe a statistical framework that encompasses many of the analytical goals in gene expression analysis; our framework is completely compatible with many of the current approaches and, in fact, can increase their utility. We propose the use of a deterministic rule, applied to the parameters of the gene expression distribution, to select a target subset of genes that are of biological interest. In addition to subset membership, the target subset can include information about relationships between genes, such as clustering. This target subset presents an interesting parameter that we can estimate by applying the rule to the sample statistics of microarray data. The parametric bootstrap, based on a multivariate normal model, is used to estimate the distribution of these estimated subsets and relevant summary measures of this sampling distribution are proposed. We focus on rules that operate on the mean and covariance. Using Bernstein's Inequality, we obtain consistency of the subset estimates, under the assumption that the sample size converges faster to infinity than the logarithm of the number of genes. We also provide a conservative sample size formula guaranteeing that the sample mean and sample covariance matrix are uniformly within a distance epsilon > 0 of the population mean and covariance. The practical performance of the method using a cluster-based subset rule is illustrated with a simulation study. The method is illustrated with an analysis of a publicly available leukemia data set.

Journal Article↗

Discovering causes and cures for cancer from gene expression analysis.

Tumorigenesis is governed by a series of complex genetic and epigenetic changes. Both mechanisms can result in either the silencing or aberrant expression of messages in a cell. Gene expression profiling techniques such as the serial analysis of gene expression (SAGE) or microarray analysis can provide global overviews of these changes, as well identify key genes and pathways involved in this process. This review outlines the current roles of these techniques in cancer research, and how they may contribute to finding not only mechanisms of this disease, but potential targets for therapy.

Biomarkers, Tumor↗

DNA microarray gene expression analysis of a vocal fold polyp and granuloma.

Genome-wide transcriptional profiling has important applications in advancing knowledge of vocal fold biology. With the use of DNA microarray technology, analysis of global patterns of gene expression can reveal unexpected networks of coordinated regulation in the extracellular matrix of the lamina propria. Transcriptional gene expression patterns for 2 vocal fold pathologies--vocal fold polyp (VP; N = 1) and vocal fold granuloma (VG; N = 1) were analyzed by means of DNA microarray analysis for 4,632 human genes using another patient's true vocal fold (TVF; N = 1) as a control. Twenty-four and 29 genes for VG and VP, respectively, were established to be either over- or underexpressed compared to that of TVF. Five-way cluster analysis revealed broad patterns that suggest a potential degree of organization underlying gene expression in these tissues. For the 1 VG, genes involved represent inflammation and wound healing; for the 1 VP, involved genes demonstrate a tempered wound repair response and increased epithelial manifestations. These results successfully demonstrate the use of DNA microarray technology as a new approach for further investigations dissecting vocal fold disease. Further investigation is needed on larger sample sizes to establish transcriptional gene expression patterns for VP and VG.

Adult↗

Multiplexed gene expression analysis using the invader RNA assay with MALDI-TOF mass spectrometry detection.

A mass spectrometric approach for measuring gene expression levels has been developed. This technique utilizes a signal amplification system and analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Signal amplification from the targeted RNA employs a recently developed invasive cleavage assay that does not require prior PCR amplification. The assay uses a set of target-specific probes (oligonucleotides), which hybridize to the RNA being measured to create an overlap structure with a single-stranded flap. This flap is enzymatically cleaved and accumulates linearly in a target-specific manner. The products of the reaction, short DNA oligomers, are well suited for quantitative detection by MALDI-TOF mass spectrometry. Multiplexing is achieved by designing the assays so that reaction products for different mRNA targets have discrete masses that can be resolved in a single mass spectrum. Simultaneous analysis of human cytokine in vitro transcripts IL-1beta, TNF-alpha, and IL-6, with GAPDH as a reference standard, was used as a model system to demonstrate this novel method of gene expression analysis.

Gas Chromatography-Mass Spectrometry↗

Insights into the multistep transformation of MGUS to myeloma using microarray expression analysis.

To define specific pathways important in the multistep transformation process of normal plasma cells (PCs) to monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM), we have applied microarray analysis to PCs from 5 healthy donors (N), 7 patients with MGUS, and 24 patients with newly diagnosed MM. Unsupervised hierarchical clustering using 125 genes with a large variation across all samples defined 2 groups: N and MGUS/MM. Supervised analysis identified 263 genes differentially expressed between N and MGUS and 380 genes differentially expressed between N and MM, 197 of which were also differentially regulated between N and MGUS. Only 74 genes were differentially expressed between MGUS and MM samples, indicating that the differences between MGUS and MM are smaller than those between N and MM or N and MGUS. Differentially expressed genes included oncogenes/tumor-suppressor genes (LAF4, RB1, and disabled homolog 2), cell-signaling genes (RAS family members, B-cell signaling and NF-kappaB genes), DNA-binding and transcription-factor genes (XBP1, zinc finger proteins, forkhead box, and ring finger proteins), and developmental genes (WNT and SHH pathways). Understanding the molecular pathogenesis of MM by gene expression profiling has demonstrated sequential genetic changes from N to malignant PCs and highlighted important pathways involved in the transformation of MGUS to MM.

Cell Transformation, Neoplastic↗

Aberrant expression of the tyrosine kinase receptor EphA4 and the transcription factor twist in Sézary syndrome identified by gene expression analysis.

Sézary syndrome (Sz) is a malignancy of CD4+ memory skin-homing T cells and presents with erythroderma, lymphadenopathy, and peripheral blood involvement. To gain more insight into the molecular features of Sz, oligonucleotide array analysis was performed comparing gene expression patterns of CD4+ T cells from peripheral blood of patients with Sz with those of patients with erythroderma secondary to dermatitis and healthy controls. Using unsupervised hierarchical clustering gene, expression patterns of T cells from patients with Sz were classified separately from those of benign T cells. One hundred twenty-three genes were identified as significantly differentially expressed and had an average fold change exceeding 2. T cells from patients with Sz demonstrated decreased expression of the following hematopoietic malignancy-linked tumor suppressor genes: TGF-beta receptor II, Mxi1, Riz1, CREB-binding protein, BCL11a, STAT4, and Forkhead Box O1A. Moreover, the tyrosine kinase receptor EphA4 and the potentially oncogenic transcription factor Twist were highly and selectively expressed in T cells of patients with Sz. High expression of EphA4 and Twist was also observed in lesional skin biopsy specimens of a subset of patients with cutaneous T cell lymphomas related to Sz, whereas their expression was nearly undetectable in benign T cells or in skin lesions of patients with inflammatory dermatoses. Detection of EphA4 and Twist may be used in the molecular diagnosis of Sz and related cutaneous T-cell lymphomas. Furthermore, the membrane-bound EphA4 receptor may serve as a target for directed therapeutic intervention.

Female↗

[Cloning and expression analysis of a LIM-domain protein gene from cotton (Gossypium hirsuturm L.)].

LIM-domain protein plays an important role in various cellular processes, including construction of cytoskeleton, transcription control and signal transduction. Based on cotton fiber EST database and contig analysis, the coding region of a cotton LIM-domain protein gene (GhLIM1) was obtained by RT-PCR from 4DPA (day post anthesis) ovule with fiber. The cloned fragment of 848 bp contains an open reading frame of 570 bp, coding for a polypeptide of 189 amino acids. It was demonstrated that the deduced GhLIM1 protein was highly homologous to the LIM-domain protein of sunflower (Helianthus annuus), tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Two intact LIM-domains, with the conserved sequence of a double zinc-finger structure (C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-24-C-X2-H), were found in the GhLIM1 protein. RT-PCR and Northern blot analysis showed that GhLIM1 gene expressed in root, shoot tip, hypocotyls, bud, leaf, anther, ovule and fiber (4DPA, 12DPA, 18DPA). However it was preferentially expressed in the shoot tip, fiber and ovule. It was proposed that the express of GhLIM1 gene is related to cotton fiber development.

Amino Acid Sequence↗

Sequence, chromosomal location and expression analysis of the murine homologue of human RAD51L2/RAD51C.

The Rad51 protein has been shown to play a vital role in the DNA repair process. In humans, its interaction with proteins like BRCA1 and BRCA2 has provided an insight into the mechanism of how these molecules function as tumor suppressors. Several members of the Rad51-like family have been recently identified, including RAD51L2. This gene has been found to be amplified in breast tumors suggesting its role in tumor progression. Here, we describe the cloning of the murine homologue of the human RAD51L2/RAD51C gene. Sequence analysis has revealed that the murine Rad51l2 protein is 86% identical and 93% similar to its human homologue. In spite of such high sequence conservation, the murine protein lacks the first nine amino acids present in the human protein. We have cloned and confirmed the sequence of the 5' end of the murine Rad51l2 cDNA using 5' RACE technique as well as by sequencing the genomic region flanking the first exon of the murine Rad51l2 gene. Northern analysis shows that Rad51l2 is expressed in several adult tissues as well as in embryos at various developmental stages. The murine Rad51l2 gene maps to chromosome 11 and is located in the syntenic region of human chromosome 17q22-23, where the human RAD51L2 is present.

Animals↗