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PubMed1994

Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine1A receptor.

The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine1A (serotonin) receptor was subcloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein coupled with the glutathione S-transferase of Schistosoma japonicum. The fusion protein was purified on a glutathione-agarose affinity column and used to immunize rabbits for the production of polyclonal anti-5-hydroxytryptamine1A receptor antibodies. Enzyme-linked immunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster injection. These antibodies only marginally affected the specific binding of [3H]8-hydroxy-2-(di-n-propyl-amino) tetralin to solubilized and membrane bound 5-hydroxytryptamine1A receptors, and did not interfere with serotonin-induced inhibition of forskolin-stimulated adenylate cyclase negatively coupled to 5-hydroxytryptamine1A receptors in rat hippocampal membranes. However, antibodies were able to immunoprecipitate 5-hydroxytryptamine1A receptor binding sites solubilized from rat hippocampal membranes. The distribution of immunoautoradiographic labelling and immunohistochemical staining of rat brain sections exposed to the antibodies raised against the fusion protein superimposed to that of 5-hydroxytryptamine1A receptor binding sites labelled by specific radioligands, with marked enrichment in the limbic areas (dentate gyrus and CA1 area in the hippocampus, lateral septum, entorhinal cortex) and the anterior raphe nuclei. The differential cellular location of immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucleus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine1A receptors might differ from one neuronal cell type to another.

Adenylyl Cyclases↗

PubMed1993

The cDNA sequence encoding human protein kinase C-zeta.

A 1779-bp complementary DNA (cDNA) that encodes protein kinase C-zeta (PKC-zeta) has been isolated from a human frontal cortex library using traditional plaque-screening methods and PCR screening. The deduced 592-amino-acid sequence of the human PKC-zeta clone has a 95-96% identity to those deduced from the previously described rat and mouse PKC-zeta clones.

Amino Acid Sequence↗

PubMed1994

The genomic structure encoding human initiation factor eIF-5A.

A genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A) was isolated and its entire nucleotide sequence was determined. The whole eIF-5A coding region is not interrupted by introns. The functional eIF-5A gene is highly homologous to the corresponding complementary DNA. One single 1.4-kb transcript thereof is expressed in human cell lines. Furthermore, we also isolated and sequenced two additional eIF-5A-related sequences which are, by expression and sequence analyses, identified as pseudogenes of the functional eIF-5A. The sequence homology between these pseudogenes and the functional eIF-5A is 71 and 87%.

Base Sequence↗

PubMed1994

Primary structure of the mRNA encoding a 16.5-kDa ubiquitin-conjugating enzyme of Pisum sativum.

The complementary DNA (PsU BC4) representing an mRNA encoding an ubiquitin-conjugating enzyme (UBC) of Pisum sativum has been cloned. The coding region is 444 nucleotides (nt) in length and capable of specifying a 16.5-kDa protein of 148 amino acids (aa) with an isoelectric point of 7.95. The deduced aa sequence showed 97% identity with Arabidopsis thaliana AtUBC8-12 families and tomato ERT17, and 80% identity with yeast ScUBC4 and ScUBC5 and Drosophila melanogaster DmUBC4. The active site cysteine (Cys85) found in UBCs so far described is also conserved in the P. sativum sequence.

Amino Acid Sequence↗

PubMed1995

Cloning of a rat cDNA encoding a protein with high homology to mouse phospholipase A2-activating protein.

A complementary DNA (2510 bp) encoding a protein with high homology to mouse phospholipase A2-activating protein (PLAP) was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxic) lambda GEM-2 cDNA library. This cDNA clone, denoted as HCW9, encodes a 647-amino-acid (aa) protein with 96.1% identity in the N-terminal portion (255 aa) to mouse PLAP [Clark et al., Proc. Natl. Acad. Sci. USA 88 (1991) 5418-5422]. Four cDNA clones were further isolated by screening a primary normoxic lambda GEM-2 cDNA library with a fragment of the HCW9 clone as probe. Partial sequencing of these clones revealed that all of the four clones were identical to clone HCW9. Northern blotting analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with two different fragments of clone HCW9 as probes, demonstrated a single band with a mobility corresponding to a size of 2.5 kb, whose level was significantly decreased during chemical hypoxia.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii.

The genomic and complementary DNA sequences were determined for the unique actin-encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5'-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5'-upstream region contains four short stretches of sequence similar to the so-called 'tub box', a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism.

Actins↗

PubMed1994

Mineralocorticoid type I receptor in the rat cochlea: mRNA identification by polymerase chain reaction (PCR) and in situ hybridization.

Expression of mineralocorticoid type I receptor (MR) gene in the rat cochlea was determined using molecular biological techniques. We synthesized complementary DNA (cDNA) from rat cochlear total RNA and then amplified MR cDNA fragments by polymerase chain reaction (PCR). The amplified cDNA fragments were subcloned into an expression vector and the nucleotide sequence was analyzed to confirm the expression of mRNA encoding MR in the cochlea. We then synthesized digoxigenin-labeled riboprobes with this cloned DNA template and examined the localization of MR mRNA in the cochlea by in situ hybridization. The amino acid sequence of MR cDNA expressed in the cochlea was identical to that of the MR first cloned in the rat hippocampus. In situ hybridization showed the expression of MR mRNA in marginal cells of the stria vascularis, suggesting that aldosterone may regulate microhomeostasis of the endolymph, presumably by modulating Na, K-ATPase activity. Intense MR signal was also identified in spiral ganglion cells, the function of which remains to be determined.

Aldosterone↗

PubMed1995

Molecular cloning and chromosomal localization of a pseudogene related to the human acyl-CoA binding protein/diazepam binding inhibitor.

The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5' ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-related sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1.

Amino Acid Sequence↗

PubMed1994

Genomic organisation and functional expression of the gene encoding the human serotonin 5-HT2C receptor.

The 5-HT2C receptor gene is unique among the members of the 5-HT receptor family by virtue of its genomic organisation. The human 5-HT2C receptor gene, unlike many other genes for guanine nucleotide binding (G)-proteins, contains three introns which interrupt the coding sequence into four exons. The first two introns are at equivalent positions as compared to the intervening sequences previously found in the 5-HT2(A) receptor gene, suggesting a close evolutionary relationship between both genes. Southern blot analysis shows that the 5-HT2C receptor gene is a single copy gene. Furthermore, we report the functional expression of a complementary DNA for the 5-HT2C receptor, cloned from hippocampal RNA. Membranes prepared from NIH 3T3 cells stably expressing the 5-HT2C receptor cDNA, displayed a single population of high affinity sites for the antagonist [3H]mesulergine (Kd = 2.9 +/- 0.4 nM, Bmax = 44.3 +/- 7.2 pmol/mg protein) as well as for [3H]5-HT (Kd = 9.9 +/- 0.7 nM, Bmax = 13.6 +/- 1.0 pmol/mg protein). Displacement of [3H]mesulergine and [3H]5HT binding by ligands indicated a pharmacological similarity of these binding sites with porcine and rat choroid plexus 5-HT2C receptors. Furthermore, activation of the 5-HT2C receptor with 5-HT results in an increased phospholipase C activity.

3T3 Cells↗

PubMed1995

Enzymatic characterisation of recombinant murine inducible nitric oxide synthase.

A complementary DNA (cDNA) encoding murine inducible nitric oxide synthase was cloned from activated J774 macrophages. Expression of this cDNA in a baculovirus-insect cell system allowed comparison of the recombinant enzyme with the native homologue. Western blot analysis of activated J774 and baculovirus-infected insect cell cytosols demonstrated reactivity against a protein of 135 kDa. Kinetic studies on the recombinant and native enzymes revealed an absolute requirement for L-arginine and NADPH in order to achieve full activity. In addition, both enzymes were found to have similar maximum velocities and Km values for these two substrates. The nitric oxide synthase antagonists N-guanidino monomethyl L-arginine and N-iminoethyl L-ornithine inhibited both enzymes at a similar rate. Furthermore, comparable concentrations of inhibitor were required to achieve half maximal enzyme inhibition. These results indicate that recombinant inducible NO synthase appears to be pharmacologically indistinguishable from the native enzyme.

Amino Acid Oxidoreductases↗

PubMed2005

Cloning and cellular expression of aquaporin 9 in ovine fetal membranes.

OBJECTIVE: Amniotic fluid (AF) absorption across fetal membranes is essential for AF volume homeostasis, balancing fetal swallowing, urine flow, and lung liquid production. In sheep, AF is absorbed primarily across the amniotic membrane into fetal vasculature situated between the amnion and chorion. Aquaporins (AQPs) are cell membrane proteins that serve as water channels. Recent studies have demonstrated the expression of AQP 1, 3, 8, and 9 in human chorioamniotic membranes and placenta. As AF dynamics continued to be explored primarily in the ovine model, we sought to clone and characterize the expression of ovine AQP9 in fetal membranes. METHODS: Ovine AQP9 gene was cloned with the use of homology reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR and Northern analysis were used to determine AQP9 gene expression, and immunohistochemistry (IHC) used to localize AQP9 protein expression in ovine fetal membranes. RESULTS: A 2085-base pair (bp) full-length complementary DNA (cDNA) sequence of ovine AQP9 was cloned. The ovine AQP9 cDNA is 86%, 82%, and 82%, and the predicted amino acid sequence (295 amino acids) is 77%, 71%, and 69% identical to human, rat, and mouse AQP9, respectively. RT-PCR and Northern analysis detected AQP9 messenger RNA expression in ovine amnion and allantois, but not in placenta, chorion, or umbilical cord. Immunohistochemistry localized AQP9 protein in epithelia of amnion and allantois. CONCLUSION: The presence of significant AQP9 messenger RNA and protein expression in ovine fetal membranes suggests that AQP9 may be a major water channel for intramembranous AF resorption in sheep. The cloning of ovine AQP9 and the demonstration of AQP9 expression in amnion and allantois significantly enhances our understanding of ovine AF regulation and offers the potential for therapeutic approaches for the treatment of oligohydramnios and polyhydramnios.

Allantois↗

PubMed2004

Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus.

Pleurotolysin, a sphingomyelin-specific cytolysin consisting of A (17 kDa) and B (59 kDa) components from the basidiomycete Pleurotus ostreatus, assembles into a transmembrane pore complex. Here, we cloned complementary and genomic DNAs encoding pleurotolysin, and studied pore-forming properties of recombinant proteins. The genomic regions encoding pleurotolysin A and B contained two and eight introns, respectively, and putative promoter sequences. The complementary DNA (cDNA) for pleurotolysin A encoded 138 amino acid residues, and the predicted product was identical with natural pleurotolysin A, except for the presence of the first methionine. Recombinant pleurotolysin A lacking the first methionine was purified as a 17-kDa protein with sphingomyelin-binding activity. The cDNA for pleurotolysin B encoded a precursor consisting of 523 amino acid residues, of which N-terminal 48 amino acid residues were absent in natural pleurotolysin B. Mature and precursor forms of pleurotolysin B were expressed as insoluble 59- and 63-kDa proteins, respectively, which were unfolded with 8 M urea and refolded by 100-fold dilution with 10 mM Tris-HCl buffer, pH 8.5. Although neither recombinant pleurotolysin A nor B alone was hemolytically active at higher concentrations of up to 100 mg/ml, they cooperatively assembled into a membrane pore complex on human erythrocytes and lysed the cell as efficiently as the natural proteins at nanomolar concentrations. In contrast, the precursor of pleurotolysin B was much less hemolytically active than mature pleurotolysin B in the presence of pleurotolysin A.

Amino Acid Sequence↗

PubMed2004

Antisense protein kinase A RI alpha-induced tumor reversion: portrait of a microarray.

Antisense oligonucleotides can selectively block disease-causing genes due to the specificity of the Watson-Crick base-pairing mechanism of action. A genome-wide view of antisense technology is illustrated via protein kinase A RI alpha antisense. Complementary DNA microarray analysis of the RI alpha antisense-induced expression profile shows the up- and down-regulation of clusters of coordinately expressed genes that define the molecular portrait of a reverted tumor cell phenotype. This global view broadens the horizons of antisense technology; it advances the promise of antisense beyond a single target gene to the whole cell and the whole organism. Along with recent rapid advances in oligonucleotide technologies-including new chemical and biological understanding of more sophisticated nucleic acid drugs-oligonucleotide-based gene silencing offers not only an exquisitely specific genetic tool for exploring basic science but also an exciting possibility for treating and preventing cancer and other diseases.

Animals↗

PubMed2004

Pregnancy-associated changes in genome-wide gene expression profiles in the liver of cow throughout pregnancy.

The objective of the present study was to fabricate and use a bovine liver complementary DNA (cDNA) microarray to profile genome-wide gene expressions in the liver of cow throughout pregnancy. A cDNA library was prepared from liver total RNA collected from cows during estrous cycle and pregnancy, and from fetuses at different stages of pregnancy. The sequenced clones were compiled and annotated by basic local alignment search tool (BLASTn) and spotted onto glass slides. The annotated liver array represented 2675 genes. Of which, 1442 were known genes while 617 sequences had matches with sequences found in expressed sequence tags databases. In addition, 616 unknown sequences were found and these sequences may possibly be identified as candidates for novel bovine genes. For gene expression profiling studies, total RNA from livers of cows slaughtered on days 19, 27-28, 49-58, 150, and 245 of pregnancy (test RNAs) was separately reverse transcribed and labeled with either cyanine 5-fluorescent dye (Cy5) or Cy3. The test samples were individually compared with liver total RNA collected from nonpregnant cycling cows (control RNA) after reverse transcription and labeling with the opposite dye following a two-color hybridization method. After scanning, image acquisition, and normalization, genes that showed either more than 1.5-fold (test/control) induction or repression were selected for further analyses. Hierarchical clustering algorithm showed a clear induction of most liver genes on days 27-28 of pregnancy. Self-organizing maps algorithm identified groups of genes whose differential expression patterns were similar across pregnancy. In conclusion, we described fabrication of a bovine liver cDNA microarray, and demonstrate, for the first time, differential expression patterns of a large number of coregulated liver genes in parallel throughout pregnancy in the bovine.

Algorithms↗

PubMed2004

Comparative gene expression in the symbiotic and aposymbiotic Aiptasia pulchella by expressed sequence tag analysis.

Intracellular symbiotic relationships are prevalent between cnidarians, such as corals and sea anemones, and the photosynthetic dinoflagellate symbionts. However, there is little understanding about how the genes express when the symbiotic relationship is set up. To characterize genes involved in this association, the endosymbiosis between sea anemone, Aiptasia pulchella, and dinoflagellate zooxanthellae, Symbiodinium spp., was employed as a model. Two complementary DNA (cDNA) libraries were constructed from RNA isolated from symbiotic and aposymbiotic A. pulchella. Using single-pass sequencing of cDNA clones, a total of 870 expressed sequence tags (ESTs) clones were generated from the two libraries: 474 from symbiotic animal and 396 from aposymbiotic animal. The initial ESTs consisted of 143 clusters and 231 singletons. A BLASTX search revealed that 147 unique genes had similarities with protein sequences available from databases; 120 of these clones were categorized according to their putative function. However, many ESTs could not assign functionally. The putative roles of some of the identified genes relative to endosymbiosis were discussed. This is the first report of the use of EST analysis to examine the gene expression in symbiotic and aposymbiotic states of the cnidarians. The systematic analysis of EST from this study provides a useful database for future investigations of the molecular mechanisms involved in algal-cnidarian symbiosis.

Animals↗

PubMed2004

Synthesis and incorporation of an alpha-hexofuranosyl thymidine into oligodeoxynucleotides via its two exocyclic OH-groups.

1-(2,3-Dideoxy-3-amino-alpha-D-arabino-hexofuranosyl)thymine is considered as a conformationally restricted acyclic nucleoside using the furanose ring to link the diol backbone to the nucleobase. The appropriately substituted phosphoramidites were synthesised via 1-(5,6-di-O-acetyl-2,3-dideoxy-3-phthalimido-alpha-D-arabino-hexofuranosyl)thymine and used in oligodeoxynucleotide (ODN) synthesis. However, the binding affinity of the mixed ODNs towards complementary DNA and RNA was decreased compared to the wild-type oligos. The decrease was smaller when the monomer was inserted near the end of the sequence. The insertions into an alpha T sequence or in a beta T sequence gave nearly the same dropping in melting temperature per modification which indicates that the new nucleotide modifications behave both as alpha and beta nucleotides.

Arabinonucleosides↗

PubMed2005

Characterization and mutation analysis of the human formin-2 (FMN2) gene in women with unexplained infertility.

OBJECTIVE: Formin-2 (Fmn2) mutant mice produce oocytes with meiosis I arrest. Our aim was to describe the human FORMIN-2 (FMN2) gene and to identify DNA sequence polymorphisms in patients with unexplained infertility and multiple failed IVF cycles. DESIGN: Institutional review board-approved observational case-control study. SETTING: Infertility center and university hospital. PATIENT(S): Sixty-two fertile controls and seven subjects with unexplained infertility. INTERVENTION(S): BLASTP (www.ncbi.nlm.nih.gov) was used to map the genomic DNA and complementary DNA sequence of FMN2. Genomic DNA was extracted from blood leukocyte samples. The polymerase chain reaction was used to amplify FMN2 gene exons for analysis by denaturing gradient gel electrophoresis. MAIN OUTCOME MEASURE(S): Characterization of the FMN2 gene and identification of fragment melting polymorphisms (FMPs). RESULT(S): FMN2 includes 411,960 base pairs (bp) of DNA with 6,204 bp in 18 exons. There was no difference in FMN2 FMP allele frequencies between the controls and subjects. One patient was homozygous for one FMP. CONCLUSION(S): The human FMN2 gene is conserved between evolutionarily diverse vertebrates. It is likely that FMN2 has the same function as Fmn2 in the mouse (i.e., maintenance of the meiotic spindle). Prospective identification of patients with meiosis I arrest is necessary to determine whether FMN2 mutations are a cause of unexplained infertility.

Adult↗

PubMed2005

Utilization of mammalian cells for efficient and reliable evaluation of specificity of antibodies to unravel the cellular function of mKIAA proteins.

Complementary DNA (cDNA) clones for human KIAA genes have been isolated as long cDNAs (>4 kb) with unknown functions. To facilitate the functional analysis of these human clones, we have isolated and determined the structures of their respective mouse homologues (mKIAA genes). Furthermore, we have comprehensively raised antibodies against the translated mKIAA proteins in order to establish a platform for their functional analysis. Since the specificity of these antibodies is critical for subsequent analyses of protein function, here we introduce two assays utilizing mammalian cells to improve their evaluation. First, we have established a semi-high-throughput production of C-terminally FLAG epitope-tagged proteins for Western blotting using specially designed mammalian expression vectors. Secondly, we have utilized immunofluorescence staining of mouse cells to analyze the subcellular localization of endogenous mKIAA proteins. Importantly, these methods allow us to detect potential posttranslational modification of the mKIAA/KIAA proteins and to predict their biological function based on their subcellular localization.

Animals↗