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PubMed2002

Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans.

Pulmonary fibrosis is a progressive and largely untreatable group of disorders that affects up to 100,000 people on any given day in the United States. To elucidate the molecular mechanisms that lead to end-stage human pulmonary fibrosis we analyzed samples from patients with histologically proven pulmonary fibrosis (usual interstitial pneumonia) by using oligonucleotide microarrays. Gene expression patterns clearly distinguished normal from fibrotic lungs. Many of the genes that were significantly increased in fibrotic lungs encoded proteins associated with extracellular matrix formation and degradation and proteins expressed in smooth muscle. Using a combined set of scoring systems we determined that matrilysin (matrix metalloproteinase 7), a metalloprotease not previously associated with pulmonary fibrosis, was the most informative increased gene in our data set. Immunohistochemisry demonstrated increased expression of matrilysin protein in fibrotic lungs. Furthermore, matrilysin knockout mice were dramatically protected from pulmonary fibrosis in response to intratracheal bleomycin. Our results identify matrilysin as a mediator of pulmonary fibrosis and a potential therapeutic target. They also illustrate the power of global gene expression analysis of human tissue samples to identify molecular pathways involved in clinical disease.

Animals↗

PubMed2001

Gene expression analysis of the catalytic subunit of human telomerase (hEST2) in the differential diagnosis of serous effusions.

Diagnostic accuracy in effusion cytology based on morphologic examination is not always satisfactory. Therefore, various diagnostic adjuncts such as immunocytochemistry or deoxyribonucleic acid cytometry are employed in this diagnostic field. Recently, demonstration of telomerase activity has been proposed as a possible marker for malignancy. In this study a seminested reverse transcription-polymerase chain reaction (RT-PCR) strategy for expression analysis of the catalytic subunit of human telomerase (hEST2) was used in 58 serous effusions. RT-PCR results correlated with cytologic diagnoses in 14 of 17 malignant effusions. In eight effusions cytologically suspicious for malignancy, PCR results were in accordance with the clinical follow-up. However, hEST2 RT-PCR was also positive in six of 15 cytologically benign effusions that consisted predominantly of inflammatory and mesothelial cells. Using the telomeric repeat amplification protocol, it could be demonstrated that cultured, proliferating benign mesothelial cells may present a weak telomerase activity, as is known in other benign cells including activated lymphocytes. In conclusion, the simple and rapid method of hEST2 RT-PCR serves to support the cytologic diagnosis of malignancy, but false-positive PCR results resulting from activated lymphocytes and proliferating mesothelial cells must be considered.

Ascitic Fluid↗

PubMed2001

ZNF232: structure and expression analysis of a novel human C(2)H(2) zinc finger gene, member of the SCAN/LeR domain subfamily.

We have identified a novel zinc finger gene, ZNF232, mapped to human chromosome 17p12. The coding region of the gene is organized in three exons corresponding to a 417 amino acid long polypeptide containing a SCAN/LeR domain and five C(2)H(2)-type zinc fingers. ZNF232 is possibly a nuclear protein, as suggested by expression analysis of GFP/ZNF232 chimeric constructs. ZNF232 transcripts were detected in a wide collection of adult human tissues. The gene is possibly subjected to tissue-specific post-transcriptional regulation by means of alternative splicing.

Amino Acid Sequence↗

PubMed1994

Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer.

The gene encoding a class A beta-lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high-level amoxicillin-resistant mutant that produces large amounts of beta-lactamase (blaF*). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high-level production of the beta-lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N-terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or blaF*) to the E. coli gene phoA resulted in the production of BlaF-PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.

Alkaline Phosphatase↗

PubMed2006

Identification and expression analysis of ABC protein-encoding genes in Toxoplasma gondii. Toxoplasma gondii ATP-binding cassette superfamily.

The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii.

ATP-Binding Cassette Transporters↗

PubMed2006

Expression analysis of genes involved in fat assimilation in human monocytes.

The metabolic syndrome X is characterized by a group of risk factors such as obesity, atherogenic dyslipidemia, hypertension, and insulin resistance. To study the functional alterations resulting from genetic variations, ex vivo studies are one option to be carried out. Since it is not an easy procedure to obtain cells from the related tissues ex vivo, the aim of the present study was to investigate whether monocytes can serve as model cells. The purpose was to check if monocytes are insulin target cells or not and to elucidate the expression of genes involved in fat assimilation. Human monocytes were drawn from venous blood of healthy donors, aged 25 - 30, using density gradient separation and antibody-based magnetic cell sorting of CD14-positive cells. An expression analysis of genes was performed using RT-PCR and Western Blot. Transcripts of the three splice-variants of the Acyl-CoA binding protein (ACBP), the Medium-chain Acyl-CoA Synthetase 1 (MACS1), the Insulin Receptor (INSR) and the Peroxisome Proliferator-activated Receptor gamma (PPARgamma) are consistently expressed in monocytes of all donors. Differences in gene expression between donors are found for two other members of the MACS-family, the fatty acid transport protein 3 (FATP3) and the FATP4. On protein level, we tested for ACBP expression. The ACBP protein is consistently expressed in monocytes of all donors. Human monocytes are insulin target cells and capable of fatty acid metabolism to some extent. Ex vivo-derived monocytes could be used in additional studies for analyzing differences in genotype-dependent expression levels of genes involved in fat assimilation such as ACBP, MACS1 or PPARgamma.

Adult↗

PubMed1998

Molecular cloning and expression analysis of five novel genes in chromosome 1p36.

The human chromosome 1p36 region displays frequent nonrandom chromosomal deletions and translocations in a number of human malignancies; these are thought to inactivate tumor suppressor genes. To identify these putative tumor suppressors we employed exon trapping, cDNA selection, and zoo blot analysis to clone five new genes located in 1p36. Two of these represent novel genes and were designated C1orf1 and xylan 1,4-beta-xylosidase 1 (XBX1). Two further genes represented new members of known gene families: PTPRZ2 was a tyrosine phosphatase and FRAP2 represented a FKBP12-rapamycin-associated protein. The fifth gene identified, ENO1L1, was significantly homologous to c-myc promoter binding protein, MBP-1, and to enolase 1 (ENO1). It colocalized with alpha enolase (ENO1) on a single P1 clone. ENO1L1 differed from both ENO1 and MBP-1 in the organization of its 5' untranslated sequences. Second, MBP-1 contained two single-base insertions not present in either ENO1 or ENO1L1 sequences, which led to a shift in the MBP-1 reading frame. Expression analysis revealed two brain-specific transcripts of 7.9 and 6.5 kb for PTPRZ2. In contrast, C1orf1, FRAP2, ENO1L1, and XBX1 appeared to be expressed ubiquitously in the tissues tested, with transcript sizes of 4.5, 8.7, 1.75, and 4.5 kb, respectively. Using fluorescence in situ hybridization, we mapped the five novel genes relative to chromosome 1p36 breakpoints present in three established tumor cell lines and one nontumor cell line. The karyotypic abnormalities in these cell lines were exploited as chromosomal landmarks; we could thus show that the telomere to centromere gene order was PTPRZ2-(MBP-1/ENO1/ENO1L1)-(C1orf1/XBX1)-+ ++FRAP2. The localization of these genes to a chromosomal region that is prone to deletions in human cancers makes them potential candidate tumor suppressors.

Amino Acid Sequence↗

PubMed1991

Separate cloning and expression analysis of two protein components of 4-chlorobenzoate dehalogenase from Pseudomonas sp. C8S3.

The two protein components, II and III, of the 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 were cloned separately into Escherichia coli. Component II was obtained on plasmid pCBSII, containing a 3.0 kbp HindIII fragment, and component III on plasmid pCBSIIIb, containing a 1.3 kbp SalI/PstI fragment. The identities of the two components were confirmed by comparison with the authentic components from Pseudomonas sp. CBS3. Both components were expressed constitutively in E. coli. Neither component alone showed dehalogenating activity. Only in the mixture of crude extracts from both clones was 4-chlorobenzoate dehalogenase detectable. The specific activities in E. coli crude extracts were 2.9 mU (mg protein)-1 for component II and 3.5 mU (mg protein)-1 for component III. Expression analysis by minicell experiments revealed a single polypeptide chain of 29 kDa for component III and of 31 kDa for component III.

Adenosine Triphosphate↗

PubMed2002

Identification of cancer/testis genes by database mining and mRNA expression analysis.

Cancer/testis (CT) antigens are immunogenic proteins expressed predominantly in gametogenic tissue and cancer; they are considered promising target molecules for cancer vaccines. The identification of new CT genes is essential to the development of polyvalent cancer vaccines designed to overcome tumor heterogeneity and antigen loss. In the current study, a search for new CT genes was conducted by mining the Unigene database for gene clusters that contain expressed sequence tags derived solely from both normal testis and tumor-derived cDNA libraries. This search identified 1,325 different cancer/testis-associated Unigene clusters. The mRNA expression pattern of 73 cancer/testis-associated Unigene clusters was assessed by reverse transcriptase polymerase chain reaction. Three gene products, CT15/Hs.177959, CT16/Hs.245431 and CT17/Hs.178062, were detected only in testis and in tumor tissue. CT15 is equivalent to ADAM2/fertilin-beta. CT16, an uncharacterized gene product, has homology (30-50%) to members of the GAGE gene family and is 89% identical to CT16.2/Hs.293317, indicating that CT16 and CT16.2 are members of a new GAGE gene family. The uncharacterized gene product, CT17, has homology (30%) to phospholipase A1. RT-PCR analysis showed that CT15 is expressed exclusively in renal cancer, whereas CT16 and CT17 are expressed in a range of human cancers. Real-time RT-PCR analysis of newly defined CT genes and the prototype CT antigens, MAGE-3 and NY-ESO-1, revealed low levels (less than 3% of the level detected in testis) of CT15, CT16 and NY-ESO-1 in a limited range of normal, non-gametogenic tissues. This study demonstrates the merits of database mining with respect to the identification of tissue-restricted gene products expressed in cancer.

Antigens, Neoplasm↗

PubMed2005

Fine mapping of radiation susceptibility and gene expression analysis of LEC congenic rat lines.

LEC rats constitute an animal model of high susceptibility to X-rays. We developed congenic LEC rat lines (recipient strain, Fischer 344 (F344)) and performed genome-wide genotyping to identify radiation susceptibility genes. We mapped seven positional candidate genes, Bmp10, Gpr73, Gp9, Cnbp, Copg, Rab7, and Rpn1, to an approximately 1.2-Mb region located between loci D4Got85 and D4Got148 on chromosome 4. None of the seven genes has been reported to be associated with radiation susceptibility. Comparison of the coding sequences for these seven genes in F344 and LEC rats showed no changes in deduced amino acid sequences. We determined gene expression differences in Gp73, Gp9, and Cnbp as well as strain-specific variations in upstream sequences of these genes. Our results suggest that radiation susceptibility in the LEC rat is primarily attributable to one of the genes within this approximately 1.2-Mb region; however, expression analysis gave no clear indication as to which gene is responsible.

Animals↗

PubMed2000

Sequence and expression analysis of a surface antigen gene family of the rickettsia Anaplasma marginale.

The tick-borne rickettsial organism, Anaplasma marginale, causes a disease in cattle of world-wide economic significance. This disease, anaplasmosis, is characterized by severe hemolytic anemia, high levels of rickettsemia and, often, death in animals over 3years of age. Animals that survive acute infection remain carriers, with continuous sub-microscopic cycles of rickettsemia that can persist for the lifetime of the animal. In the search for potential recombinant immunogens, it was discovered that several surface proteins of A. marginale encode polymorphic multigene families. Despite the small size of the genome (approx. 1250kb), these surface antigen gene families comprise greater than 2% of the genome. We present here a mapping, sequencing and expression analysis of five complete or partial genes encoding MSP1b in a Florida strain of A. marginale. Two genes are complete; they encode mRNA that is translated into polypeptide products. Three genes are incomplete and appear to be derived from the complete genes by a series of segmental intragenic recombinations. In two of the incomplete genes, 5' sequence in the incomplete genes is 3' sequence in the complete genes. Recombination within these gene families may generate diversity in surface antigens through combinatorial rearrangements. This could contribute to persistence in the chronic infections caused by A. marginale and related rickettsiae.

Anaplasma↗

PubMed2005

Haemonchus contortus: molecular cloning, sequencing, and expression analysis of the gene coding for the small subunit of ribonucleotide reductase.

Gastro-intestinal (GI) parasites are of great agricultural importance, annually costing the livestock industry vast amounts in resources to control parasitism. One such GI parasite, Haemonchus contortus, is principally pathogenic to sheep; with the parasite's blood-feeding behaviour causing effects ranging from mild anaemia to mortality in young animals. Current means of control, which are dependent on repeated treatment with anthelmintic chemicals, have led to increasing drug resistance. Together with the growing concern over residual chemicals in the environment and food chain, this has led to attempts to better understand the biology of the parasite with the aim to develop alternate or supplementary means of control, including the development of molecular vaccines. As a first step towards the understanding of the synthesis of deoxyribonucleotides in H. contortus, and its potential application to therapeutic control of this economically important parasite, we report the cloning, sequencing, and mRNA expression analysis of the ribonucleotide reductase R2 gene.

Amino Acid Sequence↗

PubMed2006

Genomic array and expression analysis of frequent high-level amplifications in adenocarcinomas of the gastro-esophageal junction.

Adenocarcinomas of the gastroesophageal junction (GEJ) show frequent high-level amplifications (HLA), but the underlying genes are not well defined. We have characterized genomic gain in 14 GEJ carcinomas by array-based comparative genomic hybridization (aCGH). The most frequent gains and amplifications were detected at 7q (57%), 8q (57%), 17q (64%), and 20q (79%), with minimally amplified regions at 7q21.1, 8q24.2, 17q12, and 20q13.2. Five HLA were detected on 7q, one on 8q, two on 17q, and three on 20q. HLA of 8q24 and 17q12 were related to MYC and ERBB2, respectively. The HLA on 7q21 was associated recurrently with ABCB1, whereas the amplified region on 20q13 implicated ZNF217, BCAS1, and CYP24. RNA expression analysis of 11 adenocarcinomas by reverse-transcription polymerase chain reaction was performed for cancer-related genes residing at 7q21 (ABCB1, ABCB4, CDK6, HGF, DMTF1, SRI, TP53AP1) and 20q13 (ZNF217, BCAS1, CYP24, TNFRSF6B). The most frequently upregulated gene on 7q21 was HGF (45%), but there was no association with genomic amplification. The most frequently overexpressed gene at 20q13 was BCAS1 (27%), which was related to HLA of this region (P = 0.006) in all three cases. We conclude that HLA occur often in GEJ adenocarcinomas. The gene responsible for the HLA of 7q21 requires further investigation, whereas BCAS1 is a good candidate for the frequent amplification of 20q13.

Adenocarcinoma↗

PubMed2002

Characterisation and expression analysis of the WDR9 gene, located in the Down critical region-2 of the human chromosome 21.

We report the isolation and characterisation of the gene WDR9 (WD Repeat 9), located in the Down Syndrome critical region-2 (DCR-2) from the human chromosome 21. This gene spans 125 kb of genomic sequence and is organised in 41 exons and 40 introns. The WDR9 cDNA has a size of 13 kb and encodes for a putative protein of 2269 amino acids with a potential location in the nucleus. Expression analysis in different human adult tissues and in cultured cell lines indicates that the gene has several tissue-specific transcripts. The more significant protein signatures in the WDR9 protein sequence are for WD repeats, bromodomain, beta-ketoacyl synthases, and ribonucleoprotein (RNP). The WDR9 protein has a high similarity with the Mus musculus neuronal differentiation protein (NDRP) and a region of similarity with the region of the Yotiao protein that has been proposed to bind the NR1 subunit of the NMDA receptor. The presence of protein-protein interaction domains as such the WD repeats, and the similarity of the WDR9 protein to regulatory proteins suggest a potential involvement in some of the clinical features associated to the DCR-2.

Amino Acid Sequence↗

PubMed1993

Structure and expression analysis of a member of the human glutamate dehydrogenase (GLUD) gene family mapped to chromosome 10p11.2.

Glutamate dehydrogenase (GLUD) is a key metabolic enzyme of the mitochondrion, playing an important role in mammalian neuronal transmission. GLUD deficiency has been associated with certain forms of neurodegeneration in the human cerebellum. Genomic DNA blot hybridization analysis and identification of a large number of GLUD-specific genomic clones have suggested that human GLUD is encoded by a multigene family consisting of at least six members. A functional GLUD gene, GLUD1, has been mapped to chromosome 10q22.3-23 and a full-length "processed" GLUD gene, GLUDP1, to chromosome Xq22-23. In the context of studing the structure, the role, and the chromosomal organization of the other family members, we have analysed in detail, a cosmid clone solely reactive with the 3' region of the GLUD cDNA. Structure and expression analysis of its GLUD-specific region suggests that it represents a truncated "processed" GLUD pseudogene. Fluorescence in situ hybridization using the entire cosmid as a probe, mapped this GLUD gene locus, termed GLUDP5, to chromosome 10p11.2.

Amino Acid Sequence↗

PubMed2003

Global amplification of sense RNA: a novel method to replicate and archive mRNA for gene expression analysis.

We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA (ss cDNA). Limited PCR was used for double-stranded DNA (dsDNA) synthesis. sRNA was synthesized from PCR products by in vitro transcription (IVT). sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000- to 2500-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001-0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 5'-->3' direction; the appropriate template for any gene expression analysis.

Cell Line↗

PubMed2003

Expression analysis of a GA 20-oxidase in embryos from two sorghum lines with contrasting dormancy: possible participation of this gene in the hormonal control of germination.

The role of GAs in promoting seed germination is well known and experiments with seeds from different species have suggested the requirement of de novo synthesis of GAs upon imbibition for germination. There are also strong indications that the enhancement of GA synthesis is part of the mechanism through which environmental signals (i.e. light) induce germination. Since along the GA biosynthetic pathway, oxidation at C-20 carried out by GA 20-oxidases is thought to be a site of regulation, a cDNA clone encoding a GA 20-oxidase was isolated from embryos of sorghum (SbGA 20ox). Expression analysis of this gene in embryos within imbibed caryopses with low dormancy showed detectable amounts of the specific mRNA early upon incubation, increasing thereafter. In contrast, it remained barely detectable in embryos from dormant caryopses. Changes in endogenous GA4 levels were in agreement with those of SbGA 20ox mRNA, suggesting that GA production might be regulated differentially at the level of transcription of this gene. The expression of SbGA 20ox was enhanced in incubated embryos isolated from either type of caryopses, illustrating a physiological control exerted by the surrounding seed tissues on gene expression. The results also show that ABA leads to a suppression of transcription of this gene.

Amino Acid Sequence↗

PubMed2005

Full-length sequence and expression analysis of estrogen receptor alpha mRNA in feline mammary tumors.

We report here on the isolation and sequencing of feline estrogen receptor alpha (ER-alpha) mRNA. Feline ER-alpha showed three alternative 5' untranslated exons and a common transcript of 6183 base pairs (bp). The putative coding sequence (1788 bp) and the unusually long 3' untranslated (3'-UTR) region (4305 bp) displayed high sequence similarity with human ER-alpha. A highly conserved sequence block was found in the 3'-UTR corresponding to a putative regulatory element for mRNA stability. In addition to wild-type ER-alpha mRNA, several exon-deleted splicing variants (ERDeltaE2, ERDeltaE3, ERDeltaE4, ERDeltaE5, ERDeltaE6, ERDeltaE7) were found in various feline tissues. This is similar to what observed in human tissues and opposite to the near absence of exon-deleted isoforms in rodents. Expression analysis of exon-deleted variants was also conducted on 24 samples of feline mammary carcinoma (FMC) and 15 normal mammary gland (NMG) controls, using a "splice targeted approach". The prevalence of some variants was similar for human and feline ER-alpha, while other isoforms were expressed at different frequencies in the two species. Two of the most frequent isoforms (ERDeltaE4, ERDeltaE7) were significantly less frequent in FMCs than in NMGs, likely as a consequence of decreased expression of ER-alpha in FMCs.

5' Untranslated Regions↗