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PubMed1998

A matrix metalloproteinase gene expressed in human T lymphocytes is identical with collagenase 3 from breast carcinomas.

The response of human T lymphocytes to various stimuli includes the expression of the matrix metalloproteinase (MMP) genes stromelysin 2, gelatinase A and gelatinase B. The proteins encoded by these genes could confer the capacity to degrade macromolecular components of the extracellular matrix (ECM), and to shed transmembrane proteins such as tumor necrosis factor (TNF), TNF receptor, Interleukin-6 receptor and Fas ligand. To identify further MMP genes transcribed in T lymphocytes exposed to phorbol 12-myristate 13-acetate and a calcium ionophore, we combined reverse transcription and polymerase chain reaction using primers specific for conserved domains and detected collagenase 3 transcripts, first described in a human breast cancer. However, when the sequence of the complementary DNA was compared, additional 23 nucleotides were found in the 5' nontranslated region of the lymphocyte messenger RNA (mRNA). Northern blot analysis revealed 2 major inducible mRNA species of 1.9 and 2.8 kilobases, whose levels were lower than those of stromelysin 2. The observation that activated T lymphocytes transcribe several MMP genes, including a collagenase, indicates that the effector functions of these cells include enzymatic activities towards most constituents of the ECM, as well as some transmembrane proteins relevant to inflammation and apoptosis.

Blotting, Northern↗

PubMed2004

Cloning of apoptosis-related genes by representational difference analysis of cDNA.

Apoptosis is frequently triggered by events that alter the expression of key target genes. Under these circumstances, the genes involved can be identified by techniques that analyze gene expression. Researchers now have a choice of reliable and effective methods for differential gene expression analysis. Comparative approaches, including gene microarray analysis, serial analysis of gene expression, and differential display provide global information about expression levels. Subtractive approaches like complementary DNA representational difference analysis (cDNA RDA) and suppression subtraction polymerase chain reaction identify a focused set of differentially expressed genes. The most suitable technique to apply depends on individual circumstances. cDNA RDA is particularly useful in nonstandard model organisms for which comprehensive gene microarrays are not available and is best used for the identification of genes with a large difference in expression levels between two populations. The technique involves the generation of amplified mixtures of cDNA fragments that are typically smaller than 1000 base pairs and represent >86% of mRNA species from each starting population. Transcriptional differences between two populations can then be identified by subtraction of cDNA amplicons followed by further polymerase chain reaction amplification. The technique is capable of detecting differences for genes expressed at less than one copy per cell and is achievable using standard laboratory apparatus. cDNA RDA can identify genes not previously described in the database, can detect low abundance transcripts (e.g., from mixed cell populations), and is best applied in experiments where relatively few differentially expressed genes are expected. Here, we describe the application of cDNA RDA to the identification of apoptosis-related genes.

Animals↗

PubMed1998

Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets.

BACKGROUND: Pseudovitamin D-deficiency rickets is characterized by the early onset of rickets with hypocalcemia and is thought to be caused by a deficit in renal 25-hydroxyvitamin D3 1alpha-hydroxylase, the key enzyme for the synthesis of 1alpha,25-dihydroxyvitamin D3. METHODS: We cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase complementary DNA (cDNA) using a mouse 1alpha-hydroxylase cDNA fragment as a probe. Its genomic structure was determined, and its chromosomal location was mapped by fluorescence in situ hybridization. We then identified mutations in the 1alpha-hydroxylase gene in four unrelated patients with pseudovitamin D-deficiency rickets by DNA-sequence analysis. Both the normal and the mutant 1alpha-hydroxylase proteins were expressed in COS-1 cells and were assayed for 1alpha-hydroxylase activity. RESULTS: The gene for 25-hydroxyvitamin D3 1alpha-hydroxylase was mapped to chromosome 12q13.3, which had previously been reported to be the locus for pseudovitamin D-deficiency rickets by linkage analysis. Four different homozygous missense mutations were detected in this gene in the four patients with pseudovitamin D-deficiency rickets. The unaffected parents and one sibling tested were heterozygous for the mutations. Functional analysis of the mutant 1alpha-hydroxylase protein revealed that all four mutations abolished 1alpha-hydroxylase activity. CONCLUSIONS: Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene are a cause of pseudovitamin D-deficiency rickets.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1996

Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis.

A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.

Amino Acid Sequence↗

PubMed1991

Laminin receptor complementary DNA-deduced synthetic peptide inhibits cancer cell attachment to endothelium.

Stable attachment of cancer cells to the endothelium is a key step in the formation of metastasis. In this study, we have investigated the possibility that interaction between laminin and its Mr 67,000 high-affinity receptor (67 LR) could play a major role in this process. Scatchard analysis of laminin-binding studies showed that bovine aortic endothelial cells exhibit 46,000 high-affinity receptors that mediate, at least in part, the attachment of highly invasive melanoma cells. This endothelial cell-melanoma cell interaction was significantly inhibited by soluble laminin and by anti-laminin antibodies. Peptide G, an eicosapeptide derived from the complementary DNA sequence of the 67 LR precursor (IPCNNKGAHSVGLMWWMLAR) that specifically binds to laminin and presumably contains the active ligand-binding site of the receptor, specifically prevented attachment of the melanoma cells to both the bovine aortic endothelial cell monolayer and human umbilical vein endothelium. Thus, peptide G may selectively interfere with the metastatic cascade by inhibiting tumor cell attachment to endothelium via the laminin-67 LR pathway and is a potential new antimetastatic agent.

Amino Acid Sequence↗

PubMed2002

Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus.

To characterize genes involved in fruit body development, two complementary DNA (cDNA) libraries were constructed from RNA isolated from liquid-cultured mycelia and fruit bodies of Pleurotus ostreatus. Using single-pass sequencing of cDNA clones, 952 and 1069 expressed sequence tags (ESTs) were generated from liquid-cultured mycelia and fruit body cDNA library, respectively. A BLASTX search revealed that 390 of the liquid-cultured mycelia ESTs (41%) and 531 of the fruit body ESTs (50%) showed significant similarity to protein sequences described in the nonredudant database (E values < or =1 x 10(-5)). When liquid-cultured mycelia and fruit body ESTs were compared by the SeqMan II program, among the total of 2021 ESTs, 1256 ESTs were unigenes, and 66 unigenes (5.3%) were commonly expressed during both stages. The functional catalogs of the ESTs were made by comparison with functionally identified Saccharomyces cerevisiae genes. Liquid-cultured mycelium ESTs were compared with fruit body ESTs and changes of the expressed genes during fruit body development were analyzed.

Culture Media↗

PubMed1996

Cloning and characterization of murine endothelial constitutive nitric oxide synthase.

Complementary DNA clones encoding mouse endothelial constitutive nitric oxide synthase (ecNOS) were isolated by plaque hybridization from a murine fetal cardiac lambda ZAP II cDNA expression library using a full-length human ecNOS cDNA as the hybridization probe. DNA sequence analysis indicates a 1202 amino acid protein showing significant sequence identity with human as well as bovine ecNOS.

Amino Acid Sequence↗

PubMed1998

Cloning and characterization of the chicken thyrotropin-releasing hormone receptor.

We report on the cloning of the full-length complementary DNA for the chicken TRH receptor. Although the TRH receptor has been cloned from several mammalian species, this is the first report from another vertebrate class. The ligand binding pocket, which is situated in the transmembrane helices of the mouse and rat TRH receptors, is completely conserved in the chicken receptor. Pharmacological studies (receptor binding and signaling) employing several TRH analogs revealed that there are no significant differences between the chicken and mouse receptors. These findings show that there have been considerable evolutionary constraints on TRH receptor structure and function. Several truncated forms of the chicken TRH receptor that appear to retain a part of an intron and are truncated in the putative third intracellular loop were also cloned, but were nonfunctional. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.

Amino Acid Sequence↗

PubMed2002

Trends in genomic analysis of the cardiovascular system.

OBJECTIVE: To evaluate the opportunities afforded cardiovascular medicine by the comprehensive and integrative approaches of genomics in cellular physiology. We present a meta-analysis of recently reported results obtained by means of high-throughput technologies (complementary DNA and oligonucleotide arrays, serial analysis of gene expression [SAGE]), as well as more traditional molecular biology approaches (real-time polymerase chain reaction, differential display, and others). DATA SOURCES: Newly published articles identified on PubMed and additional data provided by authors on-line (where available). CONCLUSIONS: The impact of genomic analysis on cardiovascular research is already visible. New genes of cardiovascular interest have been discovered, while a number of known genes have been found to be changed in unexpected contexts. The patterns in the variation of expression of many genes correlate well with the models currently used to explain the pathogenesis of cardiovascular diseases. Much more work has yet to be done, however, for the full exploitation of the immense informative potential still dormant in the genomic technologies.

Cardiovascular Diseases↗

PubMed1995

Identification and characterization of a prostaglandin transporter.

Carrier-mediated prostaglandin transport has been postulated to occur in many tissues. On the basis of sequence homology, the protein of unknown function encoded by the rat matrin F/G complementary DNA was predicted to be an organic anion transporter. Expression of the matrin F/G complementary DNA in HeLa cells or Xenopus oocytes conferred the property of specific transport of prostaglandins. The tissue distribution of matrin F/G messenger RNA and the sensitivity of matrin F/G-induced prostaglandin transport to inhibitors were similar to those of endogenous prostaglandin transport. The protein encoded by the matrin F/G complementary DNA is thus preferably called PGT because it is likely to function as a prostaglandin transporter.

Amino Acid Sequence↗

PubMed2005

Cloning and biological characterization of buffalo (Bubalus bubalis) interferon-gamma.

Interferon-gamma, a major immunomodulatory cytokine, of Indian water buffalo (Bubalus bubalis) was characterized at molecular level. Complementary DNA and essential promoter region were cloned and sequenced, and functional recombinant protein was expressed in bacterial system. The cDNA has 97.8% nucleotide identity with 11-nucleotide and four-amino acid variations, and the essential promoter region has 98.4% identity with five-nucleotide variations and a four-nucleotide deletion in comparison with the corresponding bovine sequences. All the major promoter elements such as NF IL-2 like motif, cyclosporin sensitive binding element and GATA motif are strictly conserved. Recombinant buffalo-IFN-gamma expressed in bacterial system reacted with an anti-bovine-IFN-gamma monoclonal antibody in Western blot and showed antiviral activity against buffalo pox virus in cultured Madin-Darby bovine kidney (MDBK) cells by inhibiting virus induced cytopathic effect. The study shows high level sequence similarity of IFN-gamma among ruminants. In view of the immunomodulatory and antiviral activities of IFN-gamma, this molecule will be useful in better understanding of the immune system of water buffaloes.

Animals↗

PubMed2003

Molecular cloning of proopiomelanocortin (POMC) cDNA from mud turtle, Pelodiscus sinensis.

The complete complementary DNA (cDNA) of proopiomelanocortin (POMC), a common precursor of opioid hormone beta-endorphin, melanotropin (MSH), and corticortropin (ACTH), was cloned and sequenced from pituitary and hypothalamus of mud turtle (Pelodiscus sinensis) by RT-PCR and rapid amplification of cDNA end (RACE) methods. Two transcripts of POMC mRNAs with different polyadenylation sites were observed. Both transcripts had an open reading frame encoding a 261-amino acid peptide containing nine dibasic amino acids (pair of Arg and Lys), putative proteolytic cleavage sites for processing to functional peptides. All the functional peptide fragments of mud turtle POMC, gamma-MSH, alpha-MSH, ACTH, beta-MSH, and beta-endorphin, are flanked by dibasic residues as found in other tetrapods, implying that it could be processed to give rise to all members of POMC-derived peptides. The deduced amino acid sequences of mud turtle POMC displays 63-67% identity with amphibian, 59% with chicken, 48-53% with mammals, and 37-59% identity with fish. However, functional peptide fragments are much more conserved than overall sequence and intervening fragments. In addition to pituitary and brain, mud turtle POMC mRNAs are also expressed in many peripheral tissues, such as skin, thyroid, and testis. This is the first report on the complete sequence of cDNA nucleotides and deduced amino acids of POMC in reptile.

Amino Acid Sequence↗

PubMed1994

Characterization and relative abundance of alternatively spliced luteinizing hormone receptor messenger ribonucleic acid in the ovine ovary.

Complementary DNA (cDNA) clones encoding the LH receptor (LHR) were recently isolated from pig, rat, mouse, and human testes or ovaries. Many of the LHR cDNAs isolated from these species encoded incomplete and, therefore, possibly inactive forms of the LHR. The four major incomplete cDNAs, designated B, C, D, and E, were due to alternative splicing of the full-length cDNA, designated the A form. Northern analyses of messenger RNA (mRNA) encoding LHR in these species and in sheep revealed multiple mRNA species in ovarian tissue, but were unable to distinguish between the full-length (functional) form and the splice variants. We have used reverse transcription of mRNA, amplification via the polymerase chain reaction, and cDNA sequencing to determine which alternatively spliced mRNA species were present in ovine ovarian follicles and corpora lutea, and ribonuclease protection assays to confirm these results and determine the relative abundance of these splice variants. Ovine LHR cDNAs of the full-length A form, B form, and two novel splice forms, designated F and G, were isolated and sequenced. By using LHR cDNAs that spanned the regions of the gene in which the majority of splicing variation occurred, ribonuclease-protected fragments of different sizes were generated depending on which mRNA species (A-G) were present. It is estimated that the ratios of the steady state mRNA levels of the splice variant B form/full-length A form/G form/F form were 5-3.5:1:1:0.3. The E, C, and D forms were not detected, even when using the sensitive method of reverse transcription-polymerase chain reaction for the latter two forms. The overall level of expression of LHR mRNA was greater in corpora lutea than follicles, but the relative abundance of the splice variants was similar in follicles and corpora lutea.

Alternative Splicing↗

PubMed2004

Nucleotide sequence of equine erythropoietin and characterization of region-specific antibodies.

OBJECTIVE: To determine the full-length complementary DNA (cDNA) sequence of equine erythropoietin (EPO) and to develop region-specific antibodies to differentiate equine EPO (eEPO) and human EPO (hEPO). SAMPLE POPULATION: RNA and lysate extracted from renal tissues of an adult Thoroughbred. PROCEDURE: Full-length cDNA was determined by use of a reverse transcriptase-polymerase chain reaction assay and a rapid amplification of cDNA ends method. The deduced amino acid sequence was compared with sequences of EPO reported for other species. Furthermore, 4 synthetic peptides were designed in 2 distinctive parts of the eEPO and hEPO amino acid sequences to obtain antibodies specific for eEPO and hEPO. Specificity of the antibodies was tested against supernatant of homogenized equine kidney and recombinant hEPO (rhEPO) by use of western immunoblotting techniques. RESULTS: Analysis of the 1,181 bp in the nucleotide sequence revealed that eEPO was a residue of 192 amino acids. Similarity of eEPO with amino acid sequences of EPO from other species was 81.0% to 90.6%. Antibodies were specifically recognized by eEPO or rhEPO molecules. Anti-hEPO (161 to 165) antibody specifically recognized rhEPO. In contrast, anti-eEPO (133 to 144) antibody reacted with the equine kidney lysate. CONCLUSIONS AND CLINICAL RELEVANCE: We determined the cDNA and amino acid sequence of eEPO and developed region-specific antibodies that specifically recognized eEPO or rhEPO. These antibodies may be useful in distinguishing rhEPO from eEPO in a test to detect the misuse of rhEPO in racehorses.

Amino Acid Sequence↗

PubMed2001

Molecular cloning of cDNA encoding thyroid stimulating hormone beta subunit of bighead carp Aristichthys nobilis and regulation of its gene expression.

The complementary DNA (cDNA) encoding pituitary thyroid stimulating hormone beta subunit (TSH-beta) of bighead carp was cloned and regulation of its gene expression was investigated for understanding phylogenetic divergence and evolution of TSH molecule. The cDNA was obtained from bighead carp pituitary total RNA by reverse transcription and polymerase chain reaction. Oligonucleotide primers were designed from the sequence of common carp. The full length sequence was then obtained by 3' and 5' rapid amplification of cDNA ends (RACE). The full-length sequence consisting of 3' and 5' untranslated regions was 585 bp long. The predicted amino acid sequence consisted of a signal peptide of 19 amino acid residues and a mature TSH beta subunit protein of 131 residues. The coding sequences of the cDNAs showed variable percentage homologies with those of other teleosts and vertebrate species. The predicted amino acid sequence shared 71% identity with rainbow trout and salmon, 90% with goldfish, 50% with eel and 94% with common carp in the mature protein region. The percentages of identity in the same region in comparison with bovine, porcine, rat, mouse, human and chicken were only 39, 42, 41, 40, 45 and 46%, respectively. TSH beta mRNA expression was found only in the pituitary tissue out of other tissues tested as testis, muscle, brain and heart. For the first time, thyrotropin releasing hormone (TRH) and thyroxine (T4) effects on pituitary TSH mRNA expression were tested in teleosts under in vitro conditions. TRH treatment on pituitary cells increased TSH beta mRNA level, while T4 treatment decreased TSH beta mRNA level. The present study provides a direct evidence, for the first time that TRH directly upregulates TSH beta gene expression in teleosts.

Amino Acid Sequence↗

PubMed2003

Towards a catalogue of genes transcribed in multiple tissues of Mytilus galloprovincialis.

Mussels are among the most studied marine organisms because of their ecological role, economic importance and long-standing use in coastal pollution biomonitoring. Despite the bulk of data derived from traditional investigation areas, only limited knowledge is available on mussel genes and their expression in ordinary or stressing conditions. We present here the first data obtained by production and sequencing of 3' end-specific expressed sequence tags (3'ESTs) from multiple tissues of putatively unstressed mussels (Mytilus galloprovincialis). A total of 524 clusters (98 virtual consensuses and 426 singletons) derived from 829 reliable 3'ESTs were searched in the non-redundant (nr) sequence databases (National Centre for Biotechnology Information (Bethesda, USA), NCBI). Most contigs (59%) showed poor or no similarity, thus revealing unknown mussel genes, other contigs recognized some of the few available records of M. galloprovincialis (e.g. actin, cytochrome oxidase III and twitchin) and a number of significant similarities with mitochondrial (mt) or nuclear genes from other organisms were found. Actually, a variety of ribosomal as well as motility- and adhesion-related genes and some genes potentially involved in stress responses (e.g. myticin A, heat shock proteins, methallothionein) were putatively identified. Uniquely, mitochondrial transcripts were primarily represented by a cluster of 123 3'ESTs (1296 base pairs (bp) of the mussel 16S rRNA). In our knowledge, these results provide the first systematic production and annotation of ESTs in M. galloprovincialis. They also represent the first report of a wider project, based on the strategy of 3'EST identification from normal and stressed mussels and intended to define a mussel complementary DNA (cDNA) microarray for genome-wide transcription studies.

Animals↗

PubMed2004

cDNA cloning, sequence analysis and expression of 3-methylcholanthrene-inducible cytochrome P450 1B1 in carp (Cyprinus carpio).

Cytochrome P450 (CYP) genes, which make up a large gene superfamily, are known to play an important role in drug metabolism. The levels of expression of CYP genes in the tissue of fish inhabiting polluted areas have been used extensively in biomonitoring studies as indicators of dioxin pollution. Complementary DNA of cytochrome CYP1B1 was isolated from carp (Cyprinus carpio) liver 24 h after the injection of 3-methylcholanthrene (3-MC). The full length cDNA obtained contained a 5' noncoding region of 178 bp, an open reading frame of 1593 bp coding for 530 amino acids and a stop codon, and a 3' noncoding region of 1599 bp. The predicted molecular weight of the encoded protein was approximately 60.2 kDa. The deduced amino acid sequence exhibited an identity of 60.6% with reported CYP1B sequences of plaice CYP1B, and of 52.4, 51.4, and 50.3% with human, rat, and mouse CYP1B1s, respectively. It exhibited similarities of 48.4 and 47.3% with scup CYP1C2 and -1C1 sequences. The percent identities with CYP1A sequences showed lower values in the range from 35.3 to 39.5% with mammals and teleosts. The phylogenetic tree of the CYP1 family members constructed by the protein maximum likelihood method indicates that the carp CYP1B1 and plaice CYP1B share a common ancestry with the mammalian CYP1B1s. Carp treated with 3-MC showed expression of CYP1B1 in liver, intestine and gills with distinct induction except for the gills that showed marked constitutive expression. The presence of two successive signals in treated gills at low stringency hybridization may suggest the existence of another CYP1B member that is expressed in the gills of carp.

Animals↗

PubMed2000

Cloning and sequence analysis of cDNA for diuretic hormone receptor from the Bombyx mori.

The insect diuretic hormone (DH) binds to their receptor in malpighian tubules, and stimulates water secretion and cAMP synthesis. Complementary DNA encoding a diuretic hormone receptor was cloned from the malpighian tubules of Bombyx mori. The cloned cDNA encodes a protein consisting of 391 amino acid residues with the seven transmembrane domains. The receptor protein is homologous with that of other insects, and is structurally related to G-protein coupled receptors such as corticotropin relating factor (CRF), secretin, and vasoactive intestinal peptide.

Amino Acid Sequence↗