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Striated muscle preferentially expressed genes alpha and beta are two serine/threonine protein kinases derived from the same gene as the aortic preferentially expressed gene-1.

Aortic preferentially expressed gene (APEG)-1 is a 1.4-kilobase pair (kb) mRNA expressed in vascular smooth muscle cells and is down-regulated by vascular injury. An APEG-1 5'-end cDNA probe identified three additional isoforms. The 9-kb striated preferentially expressed gene (SPEG)alpha and the 11-kb SPEGbeta were found in skeletal muscle and heart. The 4-kb brain preferentially expressed gene was detected in the brain and aorta. We report here cloning of the 11-kb SPEGbeta cDNA. SPEGbeta encodes a 355-kDa protein that contains two serine/threonine kinase domains and is homologous to proteins of the myosin light chain kinase family. At least one kinase domain is active and capable of autophosphorylation. In the genome, all four isoforms share the middle three of the five exons of APEG-1, and they differ from each other by using different 5'- and 3'-ends and alternative splicing. We show that the expression of SPEGalpha and SPEGbeta is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiation in vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEGalpha and SPEGbeta can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.

Amino Acid Sequence↗

A high proportion of polymorphisms in the promoters of brain expressed genes influences transcriptional activity.

There is increasing interest in the possibility that polymorphisms affecting gene expression are responsible for a significant proportion of heritable human phenotypic variation, including human disease. We have sought to determine if polymorphisms in the promoters of brain expressed genes are commonly functional. We screened for polymorphism 56 genes previously reported to be differentially expressed in the brains of schizophrenics [Y. Hakak, J.R. Walker, C. Li, W.H. Wong, K.L. Davis, J.D. Buxbaum, V. Haroutunian, A.A. Fienberg, Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc. Natl. Acad. Sci. 98 (2001) 4746-4751.]. We found 60 variants distributed across 31 of the genes. A total of 77 haplotypes representing 28 different putative promoters were analyzed in a reporter gene assay in two cell lines. Of a total of 54 sequence variants represented in the haplotypes, 12 (or around 22%) were functional according to a highly conservative definition. These were found in the promoters of eight genes: NPY, PCSK1, NEFL, KIAA0513, LMO4, HSPA1B, TF and MDH1. We therefore estimate that around 20-25% of promoter polymorphisms in brain expressed genes are functional, and this is likely to be an underestimate. Our data therefore provide for the first time empirical evidence that promoter element polymorphisms, at least in brain expressed genes, should be afforded a high priority for molecular genetic studies.

Base Sequence↗

Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonads.

Improved tools for Pseudomonas research include small, broad-host-range vectors that allow regulated expression from the lac operon and T7 promoters whose biology is well understood and adaptable to many bacteria. To facilitate studies on gene regulation, tracking and monitoring of bacteria in diverse environments, and the construction of biosensors, various reporter genes with versatile assay formats have been developed that can be delivered on plasmid, transposon and integration-proficient vectors.

Bacterial Proteins↗

The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH.

The pH regulation of gene expression in Aspergillus nidulans is mediated by pacC, whose 678 residue-derived protein contains three putative Cys2His2 zinc fingers. Ten pacCc mutations mimicking growth at alkaline pH remove between 100 and 214 C-terminal residues, including a highly acidic region containing an acidic glutamine repeat. Nine pacC+/- mutations mimicking acidic growth conditions remove between 299 and 505 C-terminal residues. Deletion of the entire pacC coding region mimics acidity but leads additionally to poor growth and conidiation. A PacC fusion protein binds DNA with the core consensus GCCARG. At alkaline ambient pH, PacC activates transcription of alkaline-expressed genes (including pacC itself) and represses transcription of acid-expressed genes. pacCc mutations obviate the need for pH signal transduction.

Acid-Base Equilibrium↗

Retinal selectivity of gene expression in rat retinal versus brain capillary endothelial cell lines by differential display analysis.

PURPOSE: The retina is a neural tissue especially differentiated for vision and, thus, the inner blood-retinal barrier (inner BRB) specific molecules may play an essential role in maintaining neural functions in the retina. The purpose of the present study was to identify selectively expressed genes at the inner blood-retinal barrier compared with the blood-brain barrier (BBB). METHODS: A comparison of expressed genes between conditionally immortalized rat retinal (TR-iBRB) cell lines and brain capillary endothelial (TR-BBB) cell lines was performed using mRNA differential display analysis and quantitative real time PCR analysis. The rat M-cadherin gene was cloned by performing 5' RACE, and its protein expression was detected by immunoblot analysis. RESULTS: Eight clones were identified as highly expressed genes in TR-iBRB cells including GATA-binding protein-3 (GATA-3), cytosolic branched chain amino transferase (BCATc), and M-cadherin (cadherin-15). The rat M-cadherin gene was cloned from TR-iBRB cells, for the first time, and has >86% amino acid sequence identity to the previously cloned mammalian M-cadherins. Rat M-cadherin expression in TR-iBRB cells was much greater than that in TR-BBB cells as far as mRNA and protein levels were concerned. CONCLUSIONS: M-cadherin, GATA-3, and BCATc are highly expressed in TR-iBRB cells compared with TR-BBB cells and may indeed be involved in unique functions at the inner BRB.

Amino Acid Sequence↗

A comparative analysis by SAGE of gene expression profiles of Barrett's esophagus, normal squamous esophagus, and gastric cardia.

BACKGROUND & AIMS: The metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar-lined epithelium, known as Barrett's esophagus (BE), is poorly understood. The aim of this study was to define, analyze, and compare transcription profiles of BE, normal cardia epithelium, and squamous epithelium to gain more insight into the process of metaplasia and to identify uniquely expressed genes in these epithelia. METHODS: Serial analysis of gene expression was applied for obtaining transcription libraries of biopsy specimens taken from a BE-affected patient with intestinal type of metaplasia and from normal squamous and gastric cardia epithelia. Validation of results by reverse-transcription polymerase chain reaction and immunoblotting was performed using tissues of 20 patients with BE. RESULTS: More than 120,000 tags were sequenced. Between BE and squamous 776, and between BE and gastric cardia 534 tags were significantly differentially expressed (P < .05, pairwise comparison). In contrast, squamous compared with gastric cardia epithelia showed significant differential expression of 1316 tags. The most up-regulated genes in BE compared with squamous epithelium were trefoil factors, annexin A10, and galectin-4. Each of the epithelia showed a unique cytokeratin expression profile. CONCLUSIONS: This study provides a comparison of the transcriptomes of BE, squamous epithelium, and gastric cardia epithelium. BE proves to be an incompletely differentiated type of epithelium that shows similarities to both normal squamous and gastric cardia epithelia. In addition, several uniquely expressed genes are identified. These results are a major advancement in understanding the process of metaplasia that leads to BE.

Adult↗

Identification of differentially expressed genes in oral squamous cell carcinoma.

Rapid advances in multimodality therapy have not significantly improved the overall 5-yr survival of oral cancer patients in the past two decades, thereby underscoring the need for molecular therapeutics. The development of new treatment strategies for more effective management of oral cancer requires identification of novel biological targets. Therefore, the aim of this study was to identify novel genes associated with oral tumorigenesis by comparing gene expression profile of oral squamous cell carcinomas (OSCCs) and matched nonmalignant oral epithelial tissues with differential display. Of the 180 differentially expressed cDNAs isolated, reamplified, and cloned into pGEMT-Easy Vector, 26 cDNAs were confirmed to be upregulated in OSCCs by reverse Northern blot analysis. The differentially expressed genes included components of immune system, signaling pathways, angiogenesis, cell structure, proliferation, apoptosis, cell-adhesion, and cellular metabolism. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis of 15 OSCCs and matched nonmalignant oral tissues provided the first evidence that 14-3-3-zeta, melanoma metastasizing clone D (MEMD), KIAA0471, sperm protein 17 (SP17), TC21, and anti-TNF alpha antibody are upregulated in OSCCs. Immunohistochemical analysis confirmed overexpression of 14-3-3-zeta and TC21 protein, a member of the Ras family, in OSCCs as compared to histologically normal oral tissues validating the differential display analysis. Identification of six novel differentially expressed genes in oral tumors adds to the repertoire of genes associated with oral carcinogenesis and provides candidate potential biological targets for diagnosis and/or therapy. Further characterization of the 14 unknown differentially expressed cDNAs identified in this study may provide significant clues for understanding the molecular mechanisms underlying oral tumorigenesis.

14-3-3 Proteins↗

[Detection of differentially expressed genes in head-neck carcinomas].

BACKGROUND: Carcinogenesis is generally considered a multistage process based on many alterations of the genetic substance. These alterations lead to many qualitative and quantitative changes in gene expression with subsequent malignant transformation of the cell. Only a few of these genetic alterations are identified yet. METHODS: The differential display reverse transcriptase polymerase chain reaction was employed to detect differentially expressed genes in head and neck cancer. The mRNA expression of cultivated keratinocytes of the upper aerodigestive tract was compared to the one of cultivated squamous cell carcinoma cells of the larynx, hypopharynx, and floor of the mouth. RESULTS: A total of 116 differentially expressed gene fragments were identified. They were either solely expressed by the malignant cells or the keratinocytes or in just one carcinoma cell entity. Forty-three of the 116 DNA fragments were sequenced successfully. In four cases the specific differential expression was confirmed by Northern blot hybridisation in comparison to keratinocytes, normal mucosa, and 3 benign tumors from the head and neck region. A gene bank search revealed in three cases a homology of less than 20% to already known human, animal, bacterial or viral gene sequences and in one case a homology of 98% with a human gene sequence. CONCLUSIONS: The detected for squamous cell carcinoma of the head and neck specific genes or gene fragments are the basis for further investigations to better understand carcinogenesis.

Carcinoma, Squamous Cell↗

On the mechanism by which alkaline pH prevents expression of an acid-expressed gene.

Previous work has shown that zinc finger transcription factor PacC mediates the regulation of gene expression by ambient pH in the fungus Aspergillus nidulans. This regulation ensures that the syntheses of molecules functioning in the external environment, such as permeases, secreted enzymes, and exported metabolites, are tailored to the pH of the growth environment. A direct role for PacC in activating the expression of an alkaline-expressed gene has previously been demonstrated, but the mechanism by which alkaline ambient pH prevents the expression of any eukaryotic acid-expressed gene has never been reported. Here we show that a double PacC binding site in the promoter of the acid-expressed gabA gene, encoding gamma-aminobutyrate (GABA) permease, overlaps the binding site for the transcriptional activator IntA, which mediates omega-amino acid induction. Using bacterially expressed fusion proteins, we have shown that PacC competes with IntA for DNA binding in vitro at this site. Thus, PacC repression of GABA permease synthesis is direct and occurs by blocking induction. A swap of IntA sites between promoters for gabA and amdS, a gene not subject to pH regulation, makes gabA expression pH independent and amdS acid expressed.

Alkalies↗

Differentially expressed genes that encode potential markers of goldfish macrophage development in vitro.

Primary kidney macrophage (PKM) cultures derived from goldfish hematopoietic tissues develop from early progenitors to mature macrophages in response to endogenous growth factor(s). When grown in vitro, PKM shift from a proliferative phase, where most of the proliferation and differentiation events take place, to a senescence phase, where there is cessation of proliferation and differentiation events and ultimately cell death through a process of apoptosis. The phenotypic changes of PKM from the proliferative to senescence phase are a reflection of specific changes in gene expression; therefore, comparison of gene expression patterns between the two phases should lead to the identification of macrophage genes directly involved in the positive and negative regulation of hematopoietic events, as well as genes that are modulated downstream from these regulatory points. Differential cross-screening of the proliferative phase PKM cDNA library using proliferative and senescence phase (32)P-labeled cDNA probes identified several differentially expressed genes. Specifically, initial screen of 9200 clones yielded 734 differential primary clones that were isolated and analyzed using a PCR-based secondary screen. The majority of these clone isolates encoded a single transcript as determined by PCR amplification of the primary clones. The secondary screen confirmed the differential expression of 306 clones (3.32% of the total number of screened clones). Two hundred and forty four clones were sequenced; 158 and 86 were preferentially expressed during proliferative and senescence phases, respectively. Several potential candidates of fish macrophage hematopoiesis were identified. These include, for example, zinc finger protein 147, nucleophosmin, 14-3-3 protein, adenine nucleotide translocator 2 (ANT2), granulin, survivin-1, and apoptosis inhibitor-5. In addition, several potential markers of macrophage differentiation and/or function were identified and their expression patterns characterized across three distinct stages of macrophage development in vitro. These include legumain, CD63, interferon-inducible protein, macrosialin (CD68), transcription factor MafB, and the molecular chaperone BiP/GRP78. These analyses will facilitate future characterization of macrophage developmental events by providing a more global perspective of various facets of macrophage hematopoiesis.

Animals↗

Comparative analysis for expressed genes by polymerase chain reaction using module-shuffling primers.

We have developed a method for comparative analysis of gene expression. It is based on competitive PCR amplification with module-shuffling primers, followed by gel electrophoresis in a fluorescent DNA sequencer. In this method, tagged-cDNA restriction fragments derived from different sources were amplified in one tube at the same amplification efficiency. The method can detect different amounts of each expressed gene, up to difference in amounts of 30%. The method was successfully used for comparative analysis of expressed genes in yeast.

DNA Primers↗

Computational discovery of DNA motifs associated with cell type-specific gene expression in Ciona.

Temporally and spatially co-expressed genes are expected to be regulated by common transcription factors and therefore to share cis-regulatory elements. In the ascidian Ciona intestinalis, the whole-genome sequences and genome-scale gene expression profiles allow the use of computational techniques to investigate cis-elements that control transcription. We collected 5' flanking sequences of 50 tissue-specific genes from genome databases of C. intestinalis and a closely related species Ciona savignyi. We searched for DNA motifs over-represented in upstream regions of a group of co-expressed genes. Several motifs were distributed predominantly in upstream regions of photoreceptor, pan-neuronal, or muscle-specific gene groups. One muscle-specific motif, M2, was distributed preferentially in regions from -200 to -100 bp relative to the translational start sites. Promoters of muscle-specific genes of C. intestinalis were isolated, connected with a green fluorescent protein gene (GFP), and introduced into C. intestinalis embryos. In muscle cells, these promoters specifically drove GFP expression, which mutations of the M2 sites greatly reduced. When M2 sites were located upstream of a basal promoter, the reporter GFP was specifically expressed in muscle cells. These results suggest the validity of our computational prediction of cis-regulatory elements. Thus, bioinformatics can help identify cis-regulatory elements involved in chordate development.

Animals↗

An associative analysis of gene expression array data.

MOTIVATION: We face the absence of optimized standards to guide normalization, comparative analysis, and interpretation of data sets. One aspect of this is that current methods of statistical analysis do not adequately utilize the information inherent in the large data sets generated in a microarray experiment and require a tradeoff between detection sensitivity and specificity. RESULTS: We present a multistep procedure for analysis of mRNA expression data obtained from cDNA array methods. To identify and classify differentially expressed genes, results from standard paired t-test of normalized data are compared with those from a novel method, denoted an associative analysis. This method associates experimental gene expressions presented as residuals in regression analysis against control averaged expressions to a common standard-the family of similarly computed residuals for low variability genes derived from control experiments. By associating changes in expression of a given gene to a large family of equally expressed genes of the control group, this method utilizes the large data sets inherent in microarray experiments to increase both specificity and sensitivity. The overall procedure is illustrated by tabulation of genes whose expression differs significantly between Snell dwarf mice (dw/dw) and their phenotypically normal littermates (dw/+, +/+). Of the 2,352 genes examined only 450-500 were expressed above the background levels observed in nonexpressed genes and of these 120 were established as differentially expressed in dwarf mice at a significance level that excludes appearance of false positive determinations.

Algorithms↗

Global analysis of differentially expressed genes in early gestational decidua and chorionic villi using a 9600 human cDNA microarray.

The global gene expression profiles of the decidua and chorionic villi of early human pregnancies were analysed by using cDNA microarray technology. Decidual and villous placental tissues were obtained from first trimester abortus and mRNA was extracted for cDNA microarray analysis. The human cDNA microarray [9600 clones, including known regulatory genes and expressed sequence tags (EST)] with colorimetric detection was used to identify differentially expressed genes between early gestational decidua and villi. According to cDNA microarray analysis, we have identified 641 genes with highly expressed mRNA in both decidua and villi, 49 genes with higher expressions in decidua, and 75 genes with higher expression in chorionic villi. These differentially expressed genes were further grouped into categories by their putative functions, including: cell growth-related factors, hormones/cytokines, cell adhesion molecules, signal transduction molecules, apoptosis-related factors, cytoskeleton/extracellular matrix proteins, and EST. Immunohistochemical stainings of cathepsin L, leukaemia inhibitory factor-receptor, and proliferative cell nuclear antigen showed results consistent with the microarray data. Identification of the differentially expressed genes between decidua and villi by microarray provide a global profiling of the gene expression pattern. This work adds to our understanding of placentation by reporting the gene expression profiles during first trimester human pregnancies using cDNA microarray.

Adult↗

Human globin gene expression after gene transfer.

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.

Anemia, Sickle Cell↗

[CDNA microarray on differentially expressed genes of adipose tissue in two breeds chicken].

cDNA microarray containing 9024 cDNAs was used to construct gene expression profile in order to screen differentially expressed genes of adipose tissue between broiler and Bai' er and investigate the molecular mechanism related with body fatness traits between the two breeds. Sixty seven differentially expressed genes, being involved in fat metabolism, energy metabolism, cytoskeleton, transcription and splicing factor, protein synthesis and degradation, were screened out. Furthermore, some genes that had no annotation in GenBank were screened out, they were presumed to be unknown new genes. The roles that they may play in chicken fat metabolism need clarify later.

Adipose Tissue↗

A method for analyzing the qualitative and quantitative aspects of gene expression: a transcriptional profile revealed for HeLa cells.

A number of strategies have been devised by which differentially expressed genes in different cell types or tissues can be identified. We here report an efficient method to analyze the qualitative and quantitative aspects of transcripts and to construct an extensive gene expression profile in any kind of cell or tissue of interest. This method enables us to analyze the composition of mRNA species, reflecting gene activities, by measuring the frequency of appearance of concatamerized 17mer cDNA mini-fragments, which are proportional to the abundance of mRNA. As compared with a related method previously described by others, we can analyze approximately 3-4 bp longer cDNA fragments derived from amounts of total RNA as small as 1 microg. Using this technique we examined 10 100 cDNA mini-fragments from HeLa cells and constructed a gene expression profile consisting of 3665 genes. This method should thus provide an overall indication of gene activities and a rational means for monitoring gene fluctuation in different cells or tissues at different stages of development, in normal and disease states.

Gene Expression↗