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PubMed1994

Evolution of the C4 phosphoenolpyruvate carboxylase promoter of the C4 dicot Flaveria trinervia: an expression analysis in the C3 plant tobacco.

The key enzymes of photosynthetic carbon assimilation in C4 plants have evolved from C3 isoforms which were present in the C3 ancestral species. We are interested in the molecular changes responsible for the novel expression pattern of C4 genes and are focussing on phosphoenolpyruvate carboxylase (PEPCase) of the genus Flaveria. The C4 isoform of PEPCase in the C4 plant F. trinervia is encoded by the ppcA subgroup of the PEPcase gene family and is abundantly expressed in the mesophyll cells of leaves. The orthologous ppcA genes of the C3 plant F. pringlei are only weakly expressed and their transcripts do not accumulate in a leaf-specific manner but, rather, are present in all plant organs. To answer the question whether the differences in the expression levels of the ppcA genes from F. pringlei and F. trinervia are caused by changes in the 5' upstream regions of the genes or by C4-specific trans-regulatory factors, varying parts of the 5' flanking region of the ppcA1 genes of both species were fused to the beta-glucuronidase (GUS) gene and inserted in the tobacco genome. GUS expression analysis of transgenic plants revealed that the level of expression of the Flaveria ppcA1 genes are recapitulated in the heterologous C3 plant tobacco. Hence, the 5' upstream region of the ppcA1 gene of F. trinervia contains regulatory cis-elements that are responsible for the C4-specific, abundant expression of this gene. These sequences are located upstream of position -500 relative to the transcription initiation site.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1999

Cloning of rat MLH1 and expression analysis of MSH2, MSH3, MSH6, and MLH1 during spermatogenesis.

The mismatch repair system has been highly conserved in various species. In eukaryotic cells, the Mut S and Mut L homologues play crucial roles in both DNA mismatch repair and meiotic recombination. A full-length rat cDNA clone for rat MLH1 has been constructed using the RT-PCR method. The cDNA has an open reading frame of 2274 nucleotides for a protein of 757 amino acids. We have also obtained partial cDNA clones for MSH3 and MSH6. Northern blot analysis of rat MLH1, MSH2, MSH3, and MSH6 in the testes of rats of different ages showed differential expression of these genes as a function of developmental maturation of the testes. The expression analysis suggests that MSH3 may have a more predominant role in the meiotic recombination process.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Large scale gene expression analysis of osteoclastogenesis in vitro and elucidation of NFAT2 as a key regulator.

To understand the molecular events coupling between cell proliferation and differentiation by elucidating genes essential for the process, we conducted a large scale gene expression analysis of an in vitro osteoclastogenesis system consisting of recombinant RANKL and mouse RAW264 cells. The entire process leading to the formation of tartrate resistant acid phosphatase-positive multinucleated cells takes 3 days and plates become fully covered with multinucleated cells at 4 days. Microarray probing at eight time points revealed 635 genes that showed greater than 2-fold differential expression for at least one time point and they could be classified into six groups by the "k-means" clustering analysis. Among a group of 106 early inducible genes (within 2-5 h after RANKL stimulation), four genes including NFAT2 were identified as genes whose enhanced expressions were fairly correlated with an efficient induction of matured osteoclasts. Moreover, cyclosporin A significantly suppressed the multinucleated cell formation accompanying the reduction of the nuclear localization of NFAT2. When the expression of NFAT2 was suppressed by introducing antisense NFAT2, multinucleated cell formation was severely hampered. Functional analysis thus combined with gene analysis by microarray technology elucidated a key role of NFAT2 in osteoclastogenesis in vitro.

Animals↗

PubMed2002

Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays.

Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.

DNA Probes↗

PubMed1995

Isolation and expression analysis of tyro3, a murine growth factor receptor tyrosine kinase preferentially expressed in adult brain.

Growth factors and their receptors function in the nervous system to induce proliferation and differentiation of neuronal precursor cells and to support survival of mature neurons. We have isolated a murine growth factor receptor tyrosine kinase using an anti-phosphotyrosine antibody screening procedure and studied the pattern of expression. The deduced amino acid sequence of the kinase has all the characteristics of a growth factor receptor and consists of a putative extracellular domain, a transmembrane domain, and a tyrosine kinase domain. Sequence comparison with known receptor tyrosine kinases indicated that the murine kinase is a mouse homolog of tyro3. tyro3 belongs to the Axl/Ufo growth factor receptor family. In the putative extracellular domain, there are two Ig-like domains and two fibronectin type III repeats which are conserved in other members of the Axl/Ufo family receptors. Northern blot hybridization analysis showed that tyro3 is expressed at high levels in the brain of adult mice, although considerable expression was also observed in the testis. In situ hybridization analysis revealed that high levels of tyro3 are expressed in the cerebral cortex, the lateral septum, the hippocampus, the olfactory bulb, and in the cerebellum. The highest levels of tyro3 expression in the brain are associated with neurons. The preferential expression of tyro3 in specific regions of the adult mouse brain suggests that tyro3 may function as a novel neurotrophic factor receptor.

Amino Acid Sequence↗

PubMed2000

Expression analysis of thirty one Y chromosome genes in human prostate cancer.

Rapid advances in positional cloning studies have identified most of the genes on the human Y chromosome, thereby providing resources for studying the expression of its genes in prostate cancer. Using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure, we had examined the expression of the Y chromosome genes in a panel of prostate samples diagnosed with benign prostatic hyperplasia (BPH), low and/or high grade carcinoma, and the prostatic cell line, LNCaP, stimulated by androgen treatment. Results from this expression analysis of 31 of the 33 genes, isolated so far from the Y chromosome, revealed three types of expression patterns: i) specific expression in other tissues (e.g., AMELY, BPY1, BPY2, CDY, and RBM); ii) ubiquitous expression among prostate and control testis samples, similar to those of house-keeping genes (e.g., ANT3, XE7,ASMTL, IL3RA, SYBL1, TRAMP, MIC2, DBY, RPS4Y, and SMCY); iii) differential expression in prostate and testis samples. The last group includes X-Y homologous (e.g., ZFY, PRKY, DFFRY, TB4Y, EIF1AY, and UTY) and Y-specific genes (e.g., SRY, TSPY, PRY, and XKRY). Androgen stimulation of the LNCaP cells resulted in up-regulation of PGPL, CSFR2A, IL3RA, TSPY, and IL9R and down regulation of SRY, ZFY, and DFFRY. The heterogeneous and differential expression patterns of the Y chromosome genes raise the possibility that some of these genes are either involved in or are affected by the oncogenic processes of the prostate. The up- and down-regulation of several Y chromosome genes by androgen suggest that they may play a role(s) in the hormonally stimulated proliferation of the responsive LNCaP cells.

Androgens↗

PubMed2002

Mutational and expression analysis of the reelin pathway components CDK5 and doublecortin in gangliogliomas.

Gangliogliomas represent highly differentiated glioneuronal tumors frequently occurring in young patients with focal epilepsies. Dysplastic neurons are a neuropathological hallmark of this neoplasm. Here, we have analyzed two major components of the reelin pathway associated with neuronal migration and cortical cytoarchitecture in gangliogliomas, i.e., cyclin-dependent kinase 5 (CDK5) and doublecortin (DCX). The genomic structure of human CDK5 was identified by an " in silico" cloning approach using the "high throughput genomic sequencing" (htgs) databank, NCBI BLAST 2.1. DNA sequence analysis of CDK5 and DCX was carried out in tissue samples obtained from 23 patients and compared with control DNA from non-affected individuals ( n=100). For gene expression analysis of CDK5 and DCX, a quantitative real time reverse transcription-PCR TaqMan assay was used with mRNA from gangliogliomas ( n=22) and non-lesional central nervous tissue control tissue ( n=7). The human CDK5 gene is located on chromosome 7q36 and contains 12 exons. Its coding sequence reveals 90.1% homology to the mouse counterpart. A novel pseudogene of CDK5 was found on chromosome 8. While the mutational analysis of CDK5 and DCX did not reveal any sequence alterations in gangliogliomas, a lower expression was observed for both genes in tumor compared to control tissue samples. The present data indicate that mutations of CDK5 and DCX genes are not involved in the development of gangliogliomas. A novel pseudogene on chromosome 8 has to be taken into account for future studies on CDK5.

Animals↗

PubMed1998

Cloning, expression analysis, and functional characterization of PKL12, a member of a new subfamily of ser/thr kinases.

We report the cloning of the full-length cDNA of a new murine protein kinase, mPKL12. The sequence reveals a 305-amino-acid protein that contains the characteristic subdomains of the kinase superfamily and particular homology indicating a ser/thr specificity. We have also identified its human homologue gene (94% identical) and the putative homologue proteins from Saccharomyces cerevisiae and Arabidoposis thaliana. These four sequences appear to form a new subfamily of protein kinases, close in size to the theoretical minimal catalytic domain, therefore suggesting that they could be the catalytic unit of a more complex holoenzyme. Using Escherichia coli-purified protein, we have demonstrated that the mPKL12 enzyme possesses an intrinsic kinase activity, capable of phosphorylating enolase and also of promoting autophosphorylation, with a ser/thr specificity. Tissue expression analysis of mPKL12 showed that it is ubiquitously expressed, although at very low levels. RT-PCR analysis of several cell lines also supports this view, therefore suggesting that PKL12 may play a role in a very general cellular function, probably related with the secretory pathway.

Amino Acid Sequence↗

PubMed2002

Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease.

The identification of genes that contribute to the biological basis for clinical heterogeneity and progression of prostate cancer is critical to accurate classification and appropriate therapy. We performed a comprehensive gene expression analysis of prostate cancer using oligonucleotide arrays with 63,175 probe sets to identify genes and expressed sequences with strong and uniform differential expression between nonrecurrent primary prostate cancers and metastatic prostate cancers. The mean expression value for >3,000 tumor-intrinsic genes differed by at least 3-fold between the two groups. This includes many novel ESTs not previously implicated in prostate cancer progression. Many differentially expressed genes participate in biological processes that may contribute to the clinical phenotype. One example was a strong correlation between high proliferation rates in metastatic cancers and overexpression of genes that participate in cell cycle regulation, DNA replication, and DNA repair. Other functional categories of differentially expressed genes included transcriptional regulation, signaling, signal transduction, cell structure, and motility. These differentially expressed genes reflect critical cellular activities that contribute to clinical heterogeneity and provide diagnostic and therapeutic targets.

Adult↗

PubMed2001

Cloning and expression analysis of a cDNA that encodes a leech hemerythrin.

We report the cDNA sequence of a leech hemerythrin. A cDNA was isolated from a Theromyzon tessulatum cDNA library and encodes a 120 amino acid protein of about 14 kDa. The predicted protein contains the hemerythrin signature sequence and the iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin. The protein displayed the highest identity to myohemerythrin, a non-heme iron-binding protein described in sipunculids. Expression analysis indicated that the mRNA is widely expressed in leech and is stage specific in appearance, being absent after the two first blood meals, appearing after the last blood meal during the period preceding oogenesis and disappearing after egg laying.

Amino Acid Sequence↗

PubMed2001

Systematic screening and expression analysis of the head organizer genes in Xenopus embryos.

We describe here a systematic screen of an anterior endomesoderm (AEM) cDNA library to isolate novel genes which are expressed in the head organizer region. After removing clones which hybridized to labeled cDNA probes synthesized with total RNA from a trunk region of tailbud embryos, the 5' ends of 1039 randomly picked cDNA clones were sequenced to make expressed sequence tags (ESTs), which formed 754 tentative unique clusters. Those clusters were compared against public databases and classified according to similarities found to other genes and gene products. Of them, 151 clusters were identified as known Xenopus genes, including eight organizer-specific ones (5.3%). Gene expression pattern screening was performed for 198 unique clones, which were selected because they either have no known function or are predicted to be developmental regulators in other species. The screen revealed nine possible organizer-specific clones (4.5%), four of which appeared to be expressed in the head organizer region. Detailed expression analysis from gastrula to neurula stages showed that these four genes named crescent, P7E4 (homologous to human hypothetical genes), P8F7 (an unclassified gene), and P17F11 (homologous to human and Arabidopsis hypothetical genes) demarcate spatiotemporally distinct subregions of the AEM corresponding to the head organizer region. These results indicate that our screening strategy is effective in isolating novel region-specific genes.

Amino Acid Sequence↗

PubMed1999

RT-PCR cloning, characterization and mRNA expression analysis of a cDNA encoding a type II asparagine synthetase in common bean.

Following a RT-PCR strategy based on the design of degenerate oligonucleotides resembling conserved domains of asparagine synthetase (AS; EC 6.3.5.4), we isolated a 2 kb cDNA clone (PVAS2) from root tissue of the common bean (Phaseolus vulgaris). PVAS2 encodes a protein of 584 amino acids with a predicted relative molecular mass of 65810 Da, an isoelectric point of 6.4, and a net charge of -7.2 at pH 7.0. The amino acid sequence of the protein encoded by PVAS2 is very similar to that encoded by the soybean SAS2 asparagine synthetase gene. The amino-terminal residues of the predicted PVAS2 protein are identical to the amino acids that constitute the glutamine-binding (GAT) domain of AS from other plant species, which suggests that the PVAS2 cDNA encodes a type II glutamine-dependent form of asparagine synthetase. Southern blot analysis indicates that the common bean AS is part of a small family composed of at least two genes. Expression analysis by Northern blot revealed that the PVAS2 transcript accumulates to a high level in roots and, to a lesser extent, in nodules and developing pods. Accumulation of the PVAS2 transcript in the root seems to be negatively regulated by light and sucrose, and positively regulated by nitrate.

Amino Acid Sequence↗

PubMed2000

Cloning, mapping and expression analysis of barley MADS-box genes.

Six MADS-box cDNA clones were isolated by heterologous screening from a barley inflorescence cDNA library. Based on sequence comparison to known MADS-box genes, the barley MADS-box (BM) genes were grouped into three distinct phylogenetic subclasses of the MADS-box gene family. The three MADS-box genes BM3, BM5 and BM8 share similarities with genes of the SQUAMOSA (SQUA) subgroup, while BM7 and BM9 belong to the AGAMOUS-LIKE 2 (AGL2) subgroup. BM1 resembles MADS-box genes described as solitary sequences or orphan genes. Expression analysis of the barley MADS-box genes revealed expression patterns that are not characteristic of the barley MADS-box genes of the SQUA subgroup. while expression of BM7 and BM9 was largely as expected for the AGL2 subgroup. BM1 is mainly expressed in vegetative tissues and its primary transcript undergoes alternative splicing such that the corresponding mRNAs differ by two codons. The genes BM1, BM3 and BM8 were mapped by analysis of single-nucleotide polymorphisms onto barley chromosomes 4, 2 and 7, respectively.

Alternative Splicing↗

PubMed2000

Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines.

Genetic instability is a hallmark feature of breast, colorectal and other types of cancers. One type characterized by chromosomal instability is thought to be important in the pathogenesis of many solid tumors displaying aneuploidy. Two related protein kinases and homologues of the yeast checkpoint genes, hBUB1 and hBUB1B, have been implicated in the pathogenesis of colorectal cancers. Mutations in hBUB1 have demonstrated a dominant negative effect by disrupting the mitotic checkpoint when transfected into euploid colon cancer cell lines. In Brca2 deficient murine cells, Bub1 mutants potentiate growth and cellular transformation. This would suggest that aneuploidy in solid tumors including breast, could be the result of defects in mitotic checkpoint genes and may be responsible for a chromosomal instability phenotype contributing to tumor progression. We conducted mutational analysis of 19 aneuploid breast cancer cell lines. No mutations were found but we identified nine sequence variations including five previously unreported sequence variants in hBUB1B, two of which affect restrictions sites. None of these nucleotide changes predict significant changes in the predicted protein structure. Expression analysis by Northern blot of breast cell lines showed variable expression of hBUB1 and hBUB1B genes. This suggest that while regulation of expression of these genes may be important in cancer, the lack of putative deleterious mutations in the coding sequence does not support a frequent role for mutant hBUB1 and hBUB1B alleles in the pathogenesis of breast cancer.

Alleles↗

PubMed1997

Cloning and developmental expression analysis of chick Hira (Chira), a candidate gene for DiGeorge syndrome.

Deletions within human chromosome 22q11 cause a wide variety of birth defects including the DiGeorge syndrome and velo-cardio-facial (Shprintzen) syndrome. Despite the positional cloning of several genes from the critical region, it is still not possible to state whether the phenotype is secondary to haploinsufficiency of one or more than one gene. In embryological studies phenocopies of these abnormalities are produced by a variety of actions which disrupt the contribution made by the cranial and cardiac neural crest to development. The TUPLE1/HIRA gene is related to WD40 domain transcriptional regulators and maps within the DiGeorge critical region. We have cloned the chick homologue of HIRA and conducted in situ expression analysis in early chick embryos. Hira is expressed in the developing neural plate, the neural tube, neural crest and the mesenchyme of the head and branchial arch structures. HIRA may therefore have a role in the haploinsufficiency syndromes caused by deletion of 22q11.

Amino Acid Sequence↗

PubMed2002

Long oligonucleotide arrays on nylon for large-scale gene expression analysis.

To date, most studies of multigenic expression patterns by long DNA array have used DNA fragments as probes. These probes are usually obtained as PCR products, and this represents a time-consuming and error-prone approach, requiring strict quality control. The present study examines the use of 40- and 70-mer synthetic oligonucleotides as probes for DNA array analysis with radioactive labeled targets. Design, spotting onto nylon filters, and hybridization conditions were determined and optimized. In this approach, the sensitivity and the specificity of the hybridization appear comparable to the conventional long DNA probes assay, permitting the analysis of small samples of approximately 1 microg total RNA. The long oligonucleotide array thus provides a very convenient method for the analysis of gene expression patterns in biological specimens and in clinical research.

Arabidopsis↗

PubMed2001

Protein expression analysis: from "tip of the iceberg" to a global method.

In this review I will describe the advances that have recently been made in "traditional" two-dimensional gel based protein expression analysis. A major jump has been made toward the automation of gel image analysis and comparison, one of the major bottlenecks in the analysis chain as well as the automation of spot excision and preparation for mass spectrometric analysis. Currently the gel-based "proteome mapping" approach is highly effective and 300 gels and over 10,000 spots a week can be analysed. Very recently, viable alternatives to the use of two-dimensional gel electrophoresis have emerged and these approaches are discussed here. In combination with the recently developed stable isotopic tagging methods for peptide quantitation and new mass spectrometers, this emerging technology will be a rapid and highly effective alternative to gel-based methods with few of the latter's shortcomings.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1997

The phosphoenolpyruvate carboxylase (ppc) gene family of Flaveria trinervia (C4) and F. pringlei (C3): molecular characterization and expression analysis of the ppcB and ppcC genes.

Phosphoenolpyruvate carboxylase (PEPC) is a central enzyme of C4 and CAM photosynthesis but plants, in addition, contain various non-photosynthetic isoforms with characteristic and variable functions. The partial sequence and a detailed expression analysis of the PpcB and PpcC genes which encode non-photosynthetic PEPC isoforms in the C4 plant Flaveria trinervia and the C3 species F. pringlei is presented. Southern analyses showed that PpcB and PpcC sequences are most probably of single-copy in the genomes of F. trinervia and F. pringlei. With gene-specific probes the various ppc transcripts could be distinguished unequivocally from one another and the expression patterns of all ppc genes were compared. PpcB and PpcC transcripts of both F. trinervia and F. pringlei were detected preferentially in roots and stems and at low levels in leaves. Their accumulation patterns are thus similar to each other, but different from that of the PpcA genes which in F. trinervia encode the C4 isoform of PEPC. Transgenic analysis of the 5'-flanking regions of the PpcB genes of both F. trinervia and F. pringlei in tobacco revealed that the PpcB promoter/beta-glucuronidase reporter genes were preferentially expressed in the phloem and the roots. Comparison of the PpcB promoter/reporter gene with the accumulation pattern of the PpcB transcripts in F. trinervia and F. pringlei suggests that the expression of the PpcB genes is predominantly controlled by transcription.

Base Sequence↗