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At least 307 records · Page 17Linked to original sources

PubMed2006

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

OBJECTIVE: To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. METHODS: The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. RESULTS: In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. CONCLUSION: Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed1984

Construction and characterization of a plasmid containing complementary DNA to mRNA encoding the N-terminal amino acid sequence of the rat glutathione transferase Ya subunit.

Free polyribosomal poly(A)-containing RNA isolated from normal rat liver was used to prepare a complementary DNA plasmid library in the Pst1 site of the plasmid pAT153 . A plasmid pGSTr155 complementary to mRNA coding for a glutathione transferase Ya subunit was selected by differential hybridization in situ and preliminary characterization was performed by hybrid-selected mRNA translation, immunoprecipitation and polyacrylamide-gel electrophoresis of the product synthesized in vitro. The nucleotide sequence of the complementary DNA contained within pGSTr155 was determined and shown to contain a single open reading frame corresponding to the first 129 amino acids of the N-terminus of the Ya subunit and a further 63 nucleotides upstream of the initiating methionine codon.

Amino Acid Sequence↗

PubMed2006

Regulation of corneal repair by particle-mediated gene transfer of opioid growth factor receptor complementary DNA.

OBJECTIVE: To determine whether molecular manipulation of the opioid growth factor receptor (OGFr) alters corneal reepithelialization following central corneal abrasion in rats. METHODS: The plasmid pcDNA3.1 + OGFr, carrying the rat OGFr complementary DNA in both the sense and antisense orientations, and empty vector (EV), were delivered by gene gun to the rat cornea. After 24 hours, corneas were abraded and reepithelialization was documented by fluorescein photography. Twenty-four hours after wounding, DNA synthesis (with bromodeoxyuridine) was examined. RESULTS: Eyes transfected with sense constructs of OGFr had corneal defects that were 24%, 52%, and 50% larger than the EV group at 16, 24, and 28 hours, respectively. Conversely, corneas transfected with antisense constructs of OGFr had corneal defects that were 56% and 48% smaller than the EV group at 16 and 24 hours, respectively. Bromodeoxyuridine labeling in the basal and suprabasal layers of the antisense group were increased 3.3- and 3.7-fold, respectively, in DNA synthesis from corresponding EV layers; DNA synthesis was comparable in the sense and EV groups. CONCLUSIONS: Excess OGFr delays reepithelialization, whereas attenuation of OGFr accelerates repair of the corneal surface. Clinical Relevance Inhibition of opioid growth factor action using gene therapy could be important in the treatment of corneal diseases such as nonhealing and recurrent erosions, diabetic keratopathy, and neurotrophic keratitis.

Animals↗

PubMed1997

Human growth hormone receptor: cloning and expression of the full-length complementary DNA after site-directed inactivation of a cryptic bacterial promoter.

Growth hormone receptor is a cytokine-type receptor which is required for normal somatic growth and for numerous metabolic processes. Its complementary DNA (cDNA) has been isolated in various species leading to intensive studies to elucidate the mechanism of action of the growth hormone. However, serious difficulties have been reported in cloning in Escherichia coli, an intact full-length human cDNA. In this study, the cDNA is shown to contain a cryptic bacterial promoter driving inappropriate expression of a part of human growth hormone (hGH) receptor which is toxic for E. coli growth. Identification of this promoter and its inactivation by changing only one nucleotide led us to obtain stable bacterial clones containing a high copy number of full-length coding sequences. This molecular clone was used in a baculovirus/insect cell system to produce large amounts of glycosylated recombinant receptor. Binding studies with 125I-labelled hGH revealed an affinity constant of 2.8 x 10(9) M(-1), similar to that reported for the native liver receptor. This report described a general method of cloning which could be applied to similar unclonable cDNA fragments.

Animals↗

PubMed1997

An expression profile of genes in human retina and isolation of a complementary DNA for a novel rod photoreceptor protein.

PURPOSE: To characterize expression patterns of active genes in human retina, and to isolate novel genes that are uniquely expressed in this tissue. METHODS: A 3'-directed complementary DNA (cDNA) library that faithfully represents the composition of messenger RNA (mRNA) was constructed with an mRNA preparation from a cadaveric human retina. A total of 925 3' terminal sequences were collected by sequencing randomly selected clones, of which 789 were regarded as representing chromosomally coded genes (gene signatures [GS]). GS were compared with each other and searched against GenBank. The resulting expression profile, listing gene species and their frequency, represents the composition of mRNA in the retina. By comparing this expression profile with those obtained from 10 other source cells or tissues, genes uniquely active in the retina were discovered, including some not previously described. A full-sized cDNA corresponding to one of these was isolated and sequenced. Its expression was analyzed by multitissue Northern hybridization and in situ hybridization to the retina specimen. It was then mapped on human chromosomes. RESULTS: In the expression profile, 108 genes were detected recurrently, suggesting that they are very active. Fifty-five of them were identified in GenBank, including the most abundant opsin gene and several other genes for phototransduction. Among the remaining novel and active genes, 19 were considered unique to retina on the basis of their representation status in other expression profiles and in dbEST. One of these was identified as a gene that encodes a novel secretory protein expressed in a rod photoreceptor that maps to chromosome 18p11.3. CONCLUSIONS: The expression profile of active genes in the retina represents the composition of mRNA, which reflects the relative activities of genes in this tissue. A comparison of this expression profile with those obtained with other tissues resulted in isolation of a novel cDNA specifically expressed in the rod photoreceptor. It is anticipated that additional novel genes that are uniquely active in the neural retina may be obtained with the same strategy, leading to further clarification of the biologic or physiological characteristics of this tissue.

Amino Acid Sequence↗

PubMed1984

Nucleotide sequence of complementary DNA and derived amino acid sequence of murine complement protein C3.

The nucleotide sequences coding for murine complement component C3 have been determined from a cloned genomic DNA fragment and several overlapping cloned complementary DNA fragments. The amino acid sequence of the protein was deduced. The mature beta and alpha subunits contain 642 and 993 amino acids respectively. Including a 24 amino acid signal peptide and four arginines in the beta-alpha transition region, which are probably not contained in the mature protein, the unglycosylated single chain precursor protein preproC3 would have a molecular mass of 186 484 Da and consist of 1663 amino acid residues. The C3 messenger RNA would be composed of a 56 +/- 2 nucleotide long 5' non-translated region, 4992 nucleotides of coding sequence, and a 3' non-translated region of 39 nucleotides, excluding the poly A tail. The beta chain contains only three cysteine residues, the alpha chain 24, ten of which are clustered in the carboxy terminal stretch of 175 amino acids. Two potential carbohydrate attachment sites are predicted for the alpha chain, none for the beta chain. From a comparison with human C3 cDNA sequence (of which over 80% has been determined) an extensive overall sequence homology was observed. Human and murine preproC3 would be of very similar length and share several noteworthy properties: the same order of the subunits in the precursor, the same basic residue multiplet in the beta-alpha transition region, and a glutamine residue in the thioester region. The equivalent position of the known factor I cleavage sites in human C3 alpha could be located in the murine C3 alpha chain and the size and sequence of the resulting peptide were deduced. A comparison of the amino acid sequences of murine C3 and human alpha 2-macroglobulin is given. Several areas of strong sequence homology are observed, and we conclude that the two genes must have evolved from a common ancestor.

Amino Acid Sequence↗

PubMed1993

Isolation of a goat acetyl-CoA carboxylase complementary DNA and effect of milking frequency on the expression of the acetyl-CoA carboxylase and fatty acid synthase genes in goat mammary gland.

1. Using the polymerase chain reaction we have isolated a partial complementary DNA for goat acetyl-CoA carboxylase which is 90 and 82% homologous to the published rat and chicken complementary DNA sequences, respectively. 2. Frequent milking causes an upregulation of the acetyl-CoA carboxylase and fatty acid synthase genes in goat mammary gland that parallels the increase in the respective enzyme activities. 3. The sequence for goat acetyl-CoA carboxylase is in the EMBL data base, Accession Number Z17803.

Acetyl-CoA Carboxylase↗

PubMed1993

Isolation of the complementary DNA encoding a mouse heparin-binding growth factor receptor with the use of a unique kinase insert sequence.

With the use of reverse transcriptase-polymerase chain reaction techniques focused on a unique kinase insert sequence, the complementary DNA for a mouse tyrosine kinase receptor gene, designated sam3, was isolated from a mouse brain complementary DNA library as a member of the heparin-binding growth factor receptor family or fibroblast growth factor receptor family. The kinase insert region was selected as the probe synthesized by polymerase chain reaction techniques because it composes a unique structure in this receptor family. The sam3 protein, 800 amino acids long, has high homology to mouse K-sam/bek (67%) and N-sam/flg (63%), which we also cloned as the mouse counterparts of human K-sam/bek and N-sam/flg genes, other members of this family. The sam3 protein also has high homology to human FGFR3 (92%) and chicken cek2 (80%) proteins. The sam3 protein is most likely to be a mouse counterpart of human FGFR3 and chicken cek2 proteins. mRNAs of K-sam/bek, N-sam/flg, and sam3/FGFR3 genes were detected in mouse embryo through some adult tissues. The relative amounts of these mRNAs were different depending on the organs examined. Thus, these gene products may have different biological functions in organ development including the central nervous system.

Amino Acid Sequence↗

PubMed1998

Human gamma-aminobutyric acid B receptor gene: complementary DNA cloning, expression, chromosomal location, and genomic organization.

BACKGROUND: The 6p21.3 region of human chromosome 6 is a genetic locus for schizophrenia, juvenile myoclonic epilepsy, and dyslexia. METHODS: Due to our interest in these disorders we performed complementary DNA (cDNA) hybridization selection on genomic DNA clones spanning this region to identify potential positional-candidate genes. RESULTS: We identified a full-length cDNA with an open reading frame of 2883 bp corresponding to a predicted protein of 961 amino acids that shares greater than 95% homology with the rat gamma-aminobutyric acid B (GABAB) receptor. Northern blot hybridization identified a 4.4-kb transcript in human brain. The human gene mapped to two sites on 6p21.3 separated by 2 Mb. Sequence analysis of both sites showed that the centromeric gene is transcribed, whereas the telomeric site is likely a pseudogene. The transcribed gene is distributed over 22 exons spanning 18 kb of genomic DNA. CONCLUSIONS: The genomic location, tissue expression, and function of the human GABAB receptor gene suggest that it is an important positional-candidate for the neurobehavioral disorders with a genetic locus on 6p21.3. In addition, delineation of the genomic organization will now permit it to be integrated as part of pharmacogenetic studies in trials of anxiolytic, narcotic, antiepileptic, and fluoxetine therapies.

Blotting, Northern↗

PubMed1987

Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila.

On the basis of electrophysiological analysis of Shaker mutants, the Shaker locus of Drosophila melanogaster has been proposed to encode a structural component of a voltage-dependent potassium channel, the A channel. Unlike sodium channels, acetylcholine receptors, and calcium channels, K+ channels have not been purified biochemically. To facilitate biochemical studies of a K+ channel, genomic DNA from the Shaker locus has been cloned. Rearrangements in five Shaker mutants have been mapped to a 60-kilobase segment of the genome. Four complementary DNA clones have been analyzed. These clones indicate that the Shaker gene contains multiple exons distributed over at least 65 kilobases of genomic DNA in the region where the mutations mapped. Furthermore, the gene may produce several classes of alternatively spliced transcripts. Two of the complementary DNA clones have been sequenced and their sequences support the hypothesis that Shaker encodes a component of a K+ channel.

Animals↗

PubMed2002

Synthesis of the DNA-[Ru(tpy)(dppz)(CH(3)CN)](2+) conjugates and their photo cross-linking studies with the complementary DNA strand.

We here report our studies on the conjugation of photoreactive Ru(2+) complex to oligonucleotides (ODNs), which give a stable duplex with the complementary target DNA strand. These functionalized DNA duplexes bearing photoreactive Ru(2+) complex can be specifically photolyzed to give the reactive aqua derivative, [Ru(tpy)(dppz)(H(2)O)](2+)-ODN (tpy = 2,2':6',2' '-terpyridine; dppz = dipyrido[3,2-a:2',3'-c]phenazine), in situ, which successfully cross-links to give photoproduct(s) in the duplex form with the target complementary DNA strand. Thus, the stable precursor of the aquaruthenium complex, the monofunctional polypyridyl ruthenium complex [Ru(tpy)(dppz)(CH(3)CN)](2+), has been site-specifically tethered to ODN, for the first time, by both solid-phase synthesis and postsynthetic modifications. (i) In the first approach, pure 3'-[Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate has been obtained in 42% overall yield (from the monomer blocks) by the automated solid-phase synthesis on a support labeled with [Ru(tpy)(dppz)Cl](+) complex with subsequent liberation of the crude conjugate from the support under mild conditions and displacement of the Cl(-) ligand by acetonitrile in the coordination sphere of the Ru(2+) label. (ii) In the second approach, the single-modified (3'- or 5'- or middle-modified) or 3',5'-bis-modified Ru(2+)-ODN conjugates were prepared in 28-50% yield by an amide bond formation between an active ester of the metal complex and the ODNs conjugated with an amino linker. The pure conjugates were characterized unambiguously by ultraviolet-visible (UV-vis) absorption spectroscopy, enzymatic digestion followed by HPLC quantitation, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry (MALDI-TOF as well as by ESI). [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODNs form highly stabilized ODN.DNA duplexes compared to the unlabeled counterpart (DeltaT(m) varies from 8.4 to 23.6 degrees C) as a result of intercalation of the dppz moiety; they undergo clean and selective photodissociation of the CH(3)CN ligand to give the corresponding aqua complex, [Ru(tpy)(dppz)(H(2)O)](2+)-ODNs (in the aqueous medium), which is evidenced from the change of their UV-vis absorption properties and the detection of the naked Ru(2+)-ODN ions generated in the course of the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. Thus, when [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate was hybridized to the complementary guanine (G)-rich target strand (T), and photolyzed in a buffer (pH 6.8), the corresponding aqua complex formed in situ immediately reacted with the G residue of the opposite strand, giving the cross-linked product. The highest yield (34%) of the photo cross-linked product obtained was with the ODN carrying two reactive Ru(2+) centers at both 3'- and 5'-ends. For ODNs carrying only one Ru(2+) complex, the yield of the cross-linked adduct in the corresponding duplex is found to decrease in the following order: 3'-Ru(2+)-ODN (22%) > 5'-Ru(2+)-ODN (9%) > middle-Ru(2+)-ODN (7%). It was also found that the photo cross-coupling efficiency of the tethered Ru(2+) complex with the target T strand decreased as the stabilization of the resulting duplex increased: 3'-Ru(2+)-ODN (VI.T) (DeltaT(m)(b) = 7 degrees C) < 5'-Ru(2+)-ODN (V.T) (DeltaT(m)(b) = 16 degrees C) < middle-Ru(2+)-ODN (VII.T) (DeltaT(m)(b) = 24.3 degrees C, Table 2). This shows that, with the rigidly packed structure, as in the duplex with middle-Ru(2+)-ODN, the metal center flexibility is considerably reduced, and consequently the accessibility of target G residue by the aquaruthunium moiety becomes severely restricted, which results in a poor yield in the cross-coupling reaction. The cross-linked product was characterized by PAGE, followed by MALDI-TOF MS.

Chromatography, High Pressure Liquid↗

PubMed1995

Cloning, sequencing, and characterization of a complementary DNA from Haematobia irritans irritans (Diptera: Muscidae) that shares significant homology to a yeast ATPase.

A study of the molecular genetics of the horn fly, Haematobia irritans irritans (L.), has resulted in the isolation and sequencing of a 2316-bp complementary DNA (cDNA) clone, HFN2b. An open reading frame was found, the deduced 464 amino acid sequence of which encodes a protein with 40% identity to AFG1, a member of a yeast family of ATPases; it contains an ATP- or GTP-binding P-loop motif. Northern and Southern blot analyses do not reveal significant differences in the expression of the gene homologous to HFN2b when comparing laboratory-reared horn flies that are susceptible or resistant to pyrethroid insecticides.

Adenosine Triphosphatases↗

PubMed1989

Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel.

The complete amino-acid sequence of the cyclic GMP-gated channel from bovine retinal rod photoreceptors, deduced by cloning and sequencing its complementary DNA, shows that the protein contains several putative transmembrane segments, followed by a region that is similar to the cyclic GMP-binding domains of cyclic GMP-dependent protein kinase. Expression of the complementary DNA produces cyclic GMP-gated channel activity in Xenopus oocytes.

Amino Acid Sequence↗

PubMed1997

Annealing of complementary DNA strands above the melting point of the duplex promoted by an archaeal protein.

One enigma in the biology of hyperthermophilic microorganisms, living near or above 100 degrees C, is how their genomes can be stable and, at the same time, plastic at temperatures above the melting point. The nonspecific DNA-binding protein Sso7d of the hyperthermophilic archaeon Sulfolobus solfataricus is known to protect DNA from thermal denaturation. We report here that Sso7d promotes the renaturation of complementary DNA strands at temperatures above the melting point of the duplex. This novel annealing activity is strictly homology-dependent, and even one mismatch in a stretch of 17 complementary bases severely reduces its efficiency. Since pairing of homologous single strands is a key step in all fundamental processes involving nucleic acids, such as transcription, replication, recombination, and repair, Sso7d is a candidate component of the protein machinery devoted to the coupling of DNA stability to metabolic flexibility at high temperature.

Archaeal Proteins↗

PubMed1999

Cloning and Sequencing of Cytochrome P450 1A Complementary DNA in Eel (Anguilla japonica).

: Cytochrome P450 1A (CYP1A) complementary DNA was isolated from eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5' untranslated region of 163 bp, an open reading flame of 1560 bp coding for 519 amino acids and a stop codon, and a 3' untranslated region of 1730 bp. The predicted molecular weight was approximately 58.4 kDa. The deduced amino acid sequence exhibited identities with reported CYP1A sequences of 80% for rainbow trout, 79% for scup, 76% for plaice and butterfly fish, and 74% for toadfish. When compared with mammalian CYP proteins, the eel CYP1A was more similar to CYP1A1 (54%-56%) than to CYP1A2 (49%-52%). Northern and Southern blot analyses showed two distinct bands, suggesting the existence of another 3-methylcholanthrene-inducible CYP1A gene in eel.

Journal Article↗

PubMed1990

Nucleotide sequence of the complementary DNA for turkey growth hormone.

Near-full length complementary DNA (cDNA) clones encoding turkey growth hormone (GH) have been isolated from a pituitary library. The longer of the two turkey GH cDNA clones that were sequenced is 803 base pairs (bp) in length and contains 41 nucleotides of the 5'-untranslated region (UTR), an open reading frame of 648 bp that encodes a 25 amino acid leader polypeptide segment as well as a 191 amino acid mature turkey GH protein, and a 3'-UTR that is 92 bp long followed by a 22 bp poly A tract. Comparison of the turkey GH nucleotide sequence to that of other avian GH clones shows the coding region to be greater than 93% homologous while the homology to mammalian GH sequences is between 68 and 78%. Northern blot analysis showed an approximate 800 bp turkey GH processed mRNA transcript that hybridized to the turkey GH cDNA probe. A large up-regulation of turkey GH transcription occurred when intact cultured pituitaries were treated with 1 nM human growth hormone releasing hormone but only modest changes were observed when cultures were treated with thyroid releasing hormone or somatostatin.

Amino Acid Sequence↗

PubMed1976

The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA.

The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.

Animals↗

PubMed1981

Cloned poliovirus complementary DNA is infectious in mammalian cells.

A complete, cloned complementary DNA copy of the RNA genome of poliovirus was constructed in the Pst I site of the bacterial plasmid pBR322. Cultured mammalian cells transfected with this hybrid plasmid produced infectious poliovirus. Cells transfected with a plasmid which lacked the first 115 bases of the poliovirus genome did not produce virus.

Animals↗