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PubMed2001

Global gene expression analysis reveals a role for the alpha 1 integrin in renal pathogenesis.

Kidney fibrosis is the hallmark of most types of progressive kidney disease, including the genetic disorder Alport's syndrome. We undertook gene expression analysis in Alport's syndrome mouse kidneys using microchip arrays to characterize the development of fibrosis. In addition to matrix and matrix-remodeling genes, consistent with interstitial fibrosis, macrophage-related genes show elevated expression levels in Alport's syndrome kidneys. Immunohistochemical analysis of kidney sections illustrated that macrophages as well as myofibroblasts accumulate in the tubular interstitium. Deletion of alpha(1) integrin results in decreased accumulation of both myofibroblasts and macrophages in the tubular interstitium in Alport's syndrome mice and delays disease progression. Transforming growth factor beta antagonism, although reducing interstitial fibrosis, does not limit macrophage accumulation in the tubular interstitium and disease progression. In this study, we identified previously overlooked inflammatory events that occur in the tubulointerstitial region. We propose that in addition to the previously suggested role for the alpha(1)beta(1) integrin in mesangial expansion and abnormal laminin deposition, this integrin may be critical for monocyte accumulation that, in turn, may lead directly to renal failure. Our gene expression and immunohistochemical data indicate that macrophage accumulation is dependent on alpha(1) integrin expression on the macrophage cell surface and that anti-alpha(1) integrin strategies may be employed as therapeutics in the treatment of chronic inflammatory and fibrotic diseases.

Animals↗

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↗

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↗

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↗

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↗

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↗

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↗

PubMed1993

Characterization and expression analysis of the murine rck gene: a protein kinase with a potential function in sensory cells.

We have characterized a murine protein kinase gene, rck, which was identified by crosshybridization with sequences from the v-ros tyrosine kinase gene under conditions of reduced stringency. cDNA analysis indicated that rck encodes a putative protein kinase related to the cdc2 subclass of the gene family and that the gene is identical to mak identified previously in the rat. An extensive expression analysis in the mouse performed by a combination of in situ hybridization and RNase protection revealed a novel and restricted pattern of expression: rck transcripts are found in two cell types involved in sensory transduction, photoreceptors and olfactory receptors as well as in epithelia of the respiratory tract and choroid plexus. Specific transcripts are also found in pre- and postmeiotic male germ cells. We suggest therefore that rck participates in signalling pathways important in a distinct set of cells, remarkably among them cells involved in sensory signal transduction.

Amino Acid Sequence↗

PubMed2000

Deletion and expression analysis of AZFa genes on the human Y chromosome revealed a major role for DBY in male infertility.

Three distinct regions, designated AZFa, b and c from proximal to distal Yq, are required for normal spermato-genesis in humans. Deletions involving AZFa (deletion interval 5C/D) seem to occur less frequently in infertile men and to be associated with a more severe testicular phenotype, with almost complete absence of germ cells. AZFa contains three genes, named USP9Y, DBY and UTY, and presents high homology with the mouse Delta Sxr (b) interval, deletion of which causes a severe spermatogenic impairment. However, the specific role of these genes in human spermatogenesis is still unknown and it is not clear which of them is responsible for the AZFa phenotype. Here we describe a complete sequence map of the AZFa region, the genomic structure of AZFa genes and their deletion analysis in a large number of infertile men characterized by well-defined spermatogenic alterations. Both USP9Y and DBY may cause severe testiculopathies, but DBY appears to be the major AZFa candidate. DBY is frequently deleted in infertile patients and its absence produces severe spermatogenic damage leading to a significant reduction of germ cells or even to their complete absence. Expression analysis of AZFa genes and their X-homologues revealed ubiquitous expression for all of them except DBY; this gene produces a long transcript which is ubiquitously expressed in addition to a shorter transcript which is only expressed in the testis, suggesting a specific role for DBY in the spermatogenic process. This hypothesis is further supported by the high similarity of DBY to other DEAD box proteins belonging to the PL10 subclass.

Animals↗

PubMed1992

Expression analysis of ruminant alpha-lactalbumin in transgenic mice: developmental regulation and general location of important cis-regulatory elements.

The bovine alpha-lactalbumin transgene with 750 bp and 336 bp of the 5' and 3' flanking region, respectively, is developmentally regulated as its endogenous counterpart in transgenic mice. Comparative expression analysis of three 5'-shortened constructs suggests that the region -477/-220 contains important cis-acting transcriptional elements. The level of expression of a long caprine alpha-lactalbumin transgene encompassing 8.5 kb and 9.5 kb of the 5' and 3' flanking region, respectively, was higher but still unrelated to the copy number. Expression of the transgenes and of endogenous milk-protein genes was tissue-specific. In contrast with a recent report, only low amounts of the relevant mRNA were detected in some skin samples, which suggests a possible contamination by mammary tissue.

Animals↗

PubMed1999

Cloning and expression analysis of a mouse gene related to Drosophila odd-skipped.

The Drosophila pair-rule gene odd-skipped (odd) and two related genes, sister of odd (sob) and bowel (bowl), encode zinc finger containing proteins, two of which play important roles in embryonic development probably functioning as transcription factors. Here we report the cloning and expression analysis of a mouse gene related to odd, odd-skipped related 1 (Osr1). During early embryogenesis Osr1 is expressed in the intermediate mesoderm and in a dynamic pattern during limb and branchial arch development.

5' Untranslated Regions↗

PubMed2000

Whole-genome expression analysis of snf/swi mutants of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Snf/Swi complex has been previously demonstrated to control transcription and chromatin structure of particular genes in vivo and to remodel nucleosomes in vitro. We have performed whole-genome expression analysis, using DNA microarrays, to study mutants deleted for a gene encoding one conserved (Snf2) or one unconserved (Swi1) Snf/Swi component. This analysis was performed on cells grown in both rich and minimal media. The microarray results, combined with Northern blot, computational, and genetic analyses, show that snf2Delta and swi1Delta mutations cause similar effects on mRNA levels, that Snf/Swi controls some genes differently in rich and minimal media, and that Snf/Swi control is exerted at the level of individual genes rather than over larger chromosomal domains. In addition, this work shows that Snf/Swi controls mRNA levels of MATalpha-specific genes, likely via controlling transcription of the regulators MATalpha1 and MCM1. Finally, we provide evidence that Snf/Swi acts both as an activator and as a repressor of transcription, and that neither mode of control is an indirect effect of the other.

Acid Phosphatase↗

PubMed2000

Characterization and expression analysis of an ancestor-type Pax gene in the hydrozoan jellyfish Podocoryne carnea.

We characterized a Pax gene from the hydrozoan Podocoryne carnea. It is most similar to cnidarian Pax-B genes and encodes a paired domain, a homeodomain and an octapeptide. Expression analysis demonstrates the presence of Pax-B transcripts in eggs, the ectoderm of the planula larva and in a few scattered cells in the apical polyp ectoderm. In developing and mature medusae, Pax-B is localized in particular endodermal cells, oriented toward the outside. Pax-B is not expressed in muscle cells. However, if isolated striated muscle tissue is activated for transdifferentiation, the gene is expressed within 1 h, before new cell types, such as smooth muscle and nerve cells, have formed. The expression data indicate that Pax-B is involved in nerve cell differentiation.

Amino Acid Sequence↗