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PubMed1998

The molecular basis for UV vision in birds: spectral characteristics, cDNA sequence and retinal localization of the UV-sensitive visual pigment of the budgerigar (Melopsittacus undulatus).

Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with lambdamax 365+/-3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.

Amino Acid Sequence↗
PubMed1996

Mouse microsomal Class 3 aldehyde dehydrogenase: AHD3 cDNA sequence, inducibility by dioxin and clofibrate, and genetic mapping.

We have cloned and sequenced the mouse AHD3 cDNA, which codes for the Class 3 microsomal aldehyde dehydrogenase (ALDH3m). The cDNA is 2,997 bp in length excluding the poly(A)+ tail, and has 5' and 3' non-translated regions of 113 bp and 1,429 bp, respectively. The deduced amino acid sequence consists of 484 amino acids, including the first methionine (Mr = 53,942), and contains a hydrophobic segment at the carboxyl terminus which is the putative membrane anchor. The mouse AHD3 protein was found to be: 95% similar to the rat microsomal ALDH3m protein, 65% identical to the mouse, rat and human cytosolic ALDH3c protein, and <28% similar to the rat Class 1 and Class 2 ALDH and methylmalonate-semialdehyde dehydrogenase proteins. Southern hybridization analysis of mouse cDNA probed with the full-length AHD3 cDNA revealed that the Ahd3 gene likely spans less than a total of 25 kb. The mouse Ahd3 gene is very tightly linked to the Ahd4 gene on chromosome 11. Mouse AHD3 mRNA levels are increased by dioxin in mouse Hepa-1c1c7 hepatoma wild-type (wt) cells but not in the Ah receptor nuclear translocator (ARNT)-defective (c4) mutant line, indicating that the induction process is mediated by the Ah (aromatic hydrocarbon) dioxin-binding receptor. AHD3 mRNA levels are also inducible by clofibrate in both the wt and c4 lines. AHD3 mRNA levels are not elevated in the CYP1A1 metabolism-deficient c37 mutant line or as part of the oxidative stress response found in the untreated 14CoS/14CoS mouse cell line. These data indicate that, although inducible by dioxin, the Ahd3 gene does not qualify as a member of the aromatic hydrocarbon [Ah] gene battery.

Aldehyde Dehydrogenase↗
PubMed1997

Characterization of the mouse beta-prime adaptin gene; cDNA sequence, genomic structure, and chromosomal localization.

Adaptins are important subunits of heterotetrameric complexes called adaptors, which participate in the clathrin-coated, vesicle-mediated endocytosis and intracellular receptor transport. The gene family of adaptins is divided into three classes, alpha, beta, and gamma, with further subdivision into beta- and beta-prime components. Two beta-prime adaptins, the rat AP105a and the human BAM22, have previously been characterized. The BAM22 gene is located on human Chromosome (Chr) 22q12 and can be considered a candidate meningioma tumor suppressor gene. We report here the characterization of the mouse ortholog of the BAM22 gene, and we suggest the name adtb1 for the mouse gene. Like the BAM22 gene, the adtb1 transcript is highly and ubiquitously expressed. We provide 3885-bp cDNA sequence, which entirely covers the open reading frame of the adtb1, capable of encoding a protein of 943 amino acids. The adtb1 protein is highly conserved (>96% identity) when compared with AP105a and BAM22 proteins. We also report the genomic organization of adtb1, which is similar to the BAM22 gene. The adtb1 gene has been assigned to mouse Chr 11, band 11A2, which confirms the synteny between human Chr 22q12 and mouse Chr 11.

Adaptor Protein Complex 1↗
PubMed1986

Amino acid sequence of the plasma membrane ATPase of Neurospora crassa: deduction from genomic and cDNA sequences.

The plasma membrane of Neurospora crassa contains an electrogenic H+-ATPase (EC 3.6.1.35), for which we have isolated and sequenced both genomic and cDNA clones. The ATPase gene is interrupted by four small introns (58-124 base pairs). It encodes a protein of 920 amino acids (Mr, 99,886) possessing as many as eight transmembrane segments. The Neurospora ATPase shows significant amino acid sequence homology with the Na+,K+- and Ca2+-transporting ATPases of animal cells, particularly in regions that appear to be involved in ATP binding and hydrolysis.

Amino Acid Sequence↗
PubMed1992

Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.

Amino Acid Sequence↗
PubMed1995

[An enzyme-linked immunosorbent assay determining anti-principal neutralizing determinant antibodies using synthetic peptides deduced from cDNA sequences of HIV-1 V3 loop domain].

The V3 domain, one of the hypervariable regions of gp 120 in HIV-1 possesses principal type-specific neutralizing determinant (PND). The V3 domain has the epitopes for class I major histocompatibility antigens of cytotoxic-T lymphocyte and helper-T-lymphocyte recognition sites, and also has major determinants for cell tropism towards macrophage, microglia, and human brain-derived fibroblast. Therefore, establishment for the assay of anti-PND antibodies in patients with HIV-1 infection appears to be important for the estimation on developing AIDS in these patients. We here describe a simple enzyme-linked immunosorbent assay (ELISA) for the measurement of anti-PND antibodies found in Japanese hemophiliacs. ELISA was performed using synthetic peptides, each 15 amino acid residues deduced from cDNA sequences of seven HIV-1 V3 domain mutants, which include 5 North-American and European strains (IIIB, MN, RF, SC, and WMJ-2) and 2 African strains (Af1. Con and Af2, Con). The specific antibody binding to each peptide was determined after subtraction of the non-specific binding from the total. In 49 control sera from healthy individuals with HIV-1 antibody negative, the absorbance (M +/- 1SD) at 405 nm was -0.002 +/- 0.064. Then, the cut off value was determined to be M + 4SD. In 48 hemophiliac sera with HIV-1 antibody negative, the absorbance was uniformly less than the cut off value. However, among 44 hemophiliac sera with HIV-1 antibody positive, the anti-PND antibodies were detected in the following frequencies; 2 for IIIB, 20 for MN, 1 for RF, 1 for Af1. Con, and 5 for Af2. Con.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Serodiagnosis↗
PubMed1986

cDNA sequences for human von Willebrand factor reveal five types of repeated domains and five possible protein sequence polymorphisms.

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened with two previously described cDNA inserts for human von Willebrand factor. Among 16 positive isolates, two that hybridized with a probe corresponding to the amino terminus of von Willebrand factor were sequenced. Together, these four cDNA inserts span 6.5 kilobases of the von Willebrand factor mRNA sequence, completely specifying the 2050 amino acids of the subunit of mature, secreted von Willebrand factor and 24 residues of a precursor peptide. Approximately 77% of the sequence is contained in five types of repeated domains. Domain A consists of 193-220 amino acids and is present in three tandem copies between residues 497 and 1111. Domain B contains 25-35 amino acids and is present in three copies between residues 1533 and 1636. Domain C consists of 116-119 amino acids and is duplicated between residues 1637 and 1899. In contrast to the essentially contiguous repetition of domains A-C, the two copies of domains D and E are each separated by 804 and 1383 amino acids, respectively. Domain D1 contains 289 amino acids between residues 79 and 367, while domain D2 consists of 270 amino acids between residues 1171 and 1440. Domain E1 consists of 46 amino acids between residues 25 and 70, and domain E2 consists of 46 amino acids between residues 1453 and 1498. The triplicated A domains are notably poor in Cys content, while the remaining domains are Cys-rich. The A domains appear to be homologous to a 225-residue segment of complement factor B.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Phytochrome of the green alga Mougeotia: cDNA sequence, autoregulation and phylogenetic position.

A cDNA clone encoding phytochrome (apoprotein) of the zygnematophycean green alga Mougeotia scalaris has been isolated and sequenced. The clone consisted of 3372 bp, encoded 1124 amino acids, and showed strainspecific nucleotide exchanges for M. scalaris, originating from different habitats. No indication was found of multiple phytochrome genes in Mougeotia. The 5' non-coding region of the Mougeotia PHY cDNA harbours a striking stem-loop structure. Homologies with higher-plant phytochromes were 52-53% for PHYA and 57-59% for PHYB. Highest homology scores were found with lower-plant phytochromes, for example 67% for Selaginella (Lycopodiopsida), 64% for Physcomitrella (Bryopsida) and 73% for Mesotaenium (Zygnematophyceae). In an unrooted phylogenetic tree, the position of Mougeotia PHY appeared most distant to all other known PHYs. The amino acids Gly-Val in the chromophore-binding domain (-Arg-Gly-Val-His-Gly-Cys-) were characteristic of the zygnematophycean PHYs known to date. There was no indication of a transmembrane region in Mougeotia phytochrome in particular, but a carboxyl-terminal 16-mer three-fold repeat in both, Mougeotia and Mesotaenium PHYs may represent a microtubule-binding domain. Unexpected for a non-angiosperm phytochrome, its expression was autoregulated in Mougeotia in a red/far-red reversible manner: under Pr conditions, phytochrome mRNA levels were tenfold higher than under Pfr conditions.

Amino Acid Sequence↗
PubMed1985

Molecular cloning, cDNA sequencing, and chemical synthesis of cecropin B from Hyalophora cecropia.

Two cDNA clones containing coding information for cecropin B from the Cecropia moth (Hyalophora cecropia) were identified by means of a synthetic probe. Sequencing of the two inserts showed that cecropin B is processed from a 62-amino acid residue precursor molecule including a 26-residue leader peptide and a COOH-terminal glycine residue. The latter presumably donates the nitrogen of the amide group present on the COOH-terminal leucine residue of the mature cecropin B. The sequence deduced for the mature cecropin B differed in the COOH-terminal region from the tentative structure previously determined by carboxypeptidase digestion. To settle the discrepancy, cecropin B was synthesized according to the cDNA sequence with an amidated COOH-terminal leucine. Natural and synthetic cecropin B were found to be indistinguishable with respect to electrophoretic mobility and antibacterial activity against seven different bacteria. The COOH-terminal tetrapeptides were isolated from both natural and synthetic cecropin B and found to be indistinguishable. The correct sequence for cecropin B is (formula; see text).

Amino Acid Sequence↗
PubMed1996

The complete cDNA sequence of laminin alpha 4 and its relationship to the other human laminin alpha chains.

We previously localised the gene (LAMA4) encoding a novel laminin alpha 4 chain to chromosome 6q21. In this study, we describe the complete coding sequence and compare the protein with the other three known human laminin alpha chains. Although closely linked to LAMA2, the LAMA4 product most closely resembles laminin alpha 3, a constituent of laminin 5. Like laminin alpha 3A, the alpha 4 chain is a truncated version of the alpha 1 and alpha 2 chains, with a much reduced short arm. While the alpha 4 molecule is most similar to alpha 3, it shares some features of the C-terminal domains G4 and G5 in common with alpha 2. Unlike the LAMA3 gene, LAMA4 appears to encode only a single transcript, as determined by 5' rapid amplification of cDNA ends. The cDNA sequence encodes 1816 amino acids, which include a 24-residue signal peptide. The gene is expressed in skin, placenta, heart, lung, skeletal muscle, and pancreas. We have also shown that the mRNA can be readily reverse transcribed and amplified from cultured dermal fibroblasts.

Alternative Splicing↗
PubMed2000

HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project.

HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge

Animals↗
PubMed1996

Characterization of anti-sperm antibodies and their coding cDNA sequences by Epstein-Barr virus transformed B cell lines from lymphocytes of infertile women possessing anti-sperm antibodies.

Epstein-Barr virus (EBV)-transformed B cell lines that produce human antisperm antibodies were established using peripheral blood lymphocytes (PBLs) from infertile women with sperm immobilizing antibodies in their sera. We obtained three stable cell populations (designated B1, B2, D5) of transformed PBLs originating from three different patients. They produced IgM sperm-reacting antibodies directed against the tail of live, methanol-fixed and NaIO4-treated human spermatozoa. The established antisperm antibodies recognized noncarbohydrate sperm membrane antigens with different specificity and distribution in the male reproductive system. Antisperm antibody-B2 corresponding antigen appears to be specific for the male reproductive system. This antigen is excreted from the epithelial cells of the ductus epididymidis and bound to the spermatozoa in the lumen of the ductus. Antisperm antibodies B1 and D5 corresponding antigens were expressed on the spermatozoa in the seminiferous tubules and were common to the secretions of the ductus epididymidis, prostate and some other somatic organs. The cDNA of the immunoglobulin heavy chain genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using RNA extracted from these clones. The immunoglobulin heavy chain cDNA sequences of these antisperm antibodies showed extremely high homology to previously reported immunoglobulin germline DNA sequences, implying that these antisperm antibodies might be natural autoantibodies rather than antibodies stimulated by external antigen.

Antibodies, Monoclonal↗
PubMed1994

cDNA sequence and structure of a gene encoding trout testis high-mobility-group-1 protein.

Perchloric acid extraction of trout testis nuclei revealed the presence of two large high-mobility-group (HMG) proteins, HMG-T1 and HMG-T2. The sequence of a complete cDNA (1407 bp) for trout testis HMG-1 protein (referred as to HMG-T1) has been determined. The deduced HMG-T1 protein contains 203 amino acids with more than 86% similarity to mammalian HMG-1 proteins. A single-sized mRNA for HMG-T1 has been detected by Northern-blot analysis consistent with the size derived from the HMG-T1 cDNA. Amplification of human and trout genomic DNAs by polymerase chain reaction using primers specific for trout and human HMG-1 cDNAs revealed that unlike the human genome, which contains predominantly intronless HMG-1 sequences, intronless HMG-T1 sequences were not found in the fish genome. Southern-blot analysis suggested that the trout testis HMG-1 gene is encoded by at least two sequences with high similarity. A gene encoding HMG-T1 protein has been isolated from a trout testis genomic library and by PCR of trout genomic DNA (3879 bp). The trout testis HMG-1 gene is organized into five exons (four exons corresponding to the protein-coding region) and its exon/intron boundaries are identical to those of the human HMG-2 gene [Shirakawa, H. & Yoshida, M. (1992). J. Biol. Chem. 267, 6641-6645] suggesting the evolution of HMG-1 and HMG-2 genes from a common ancestor.

Amino Acid Sequence↗
PubMed2004

Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry.

A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.

Amino Acid Sequence↗
PubMed1987

Identification of interferon-modulated proliferation-related cDNA sequences.

To identify genes mediating the antiproliferative action of interferon (IFN), two cDNA libraries were constructed with mRNA from IFN-treated and untreated human fibrosarcoma (HT1080) cells previously shown to be highly sensitive to the antiproliferative effects of IFN. Differential screening of these two libraries identified cloned sequences whose expression was either induced or repressed with IFN treatment. Rescreening of these sequences with cDNA probes constructed from proliferating or quiescent cells led to the identification of one IFN-induced and three IFN-repressed sequences whose expressions also appeared to be modulated by cell proliferation. Blot-hybridization analysis revealed that RNA levels corresponding to the three repressed genes decreased when HT1080 cells were treated with IFN or when proliferation of normal CUA foreskin fibroblast cells became naturally arrested by contact inhibition. Levels of RNA corresponding to the induced gene increased in HT1080 cells within 24 hr after IFN-treatment but declined below basal levels by 48 hr. Expression of these genes was unaffected or only slightly affected by IFN treatment in variant cells resistant to the antiproliferative effects of IFN. Collectively, these results suggest that the identified cDNAs correspond to genes that are involved in the antiproliferative action of IFN. Moreover, these results also suggest that IFN's antiproliferative action may be exerted through genes that contribute to arresting cell proliferation during contact inhibition.

Cell Division↗
PubMed2005

cDNA sequences of two biliproteins, BP1 and BP2, from the cabbage white butterfly, Pieris rapae and their tissue- and stage-specific accumulation.

Two similar full-length cDNAs of biliprotein were isolated and shown to encode the two isoelectric forms of biliverdin-binding proteins (BPs): BP1 and BP2 in Pieris rapae. Sequence analysis of two cDNA clones shows that both BPs contain a 567-bp open reading frame which predicts a 189-amino acid protein and a 15-amino acid signal peptide. The calculated isoelectric points are pI=7.25 (BP1) and 6.74 (BP2), respectively. Comparison of two sequences of BP1 and BP2 reveals 12 base differences in the open reading frame, of which three nucleotide changes lead to two amino acid substitutions. The 5'-UTR from the two clones shows no difference, but an additional 45-bp fragment is inserted in the 3'-UTR of BP2 making its message a little longer than that of BP1. Northern blot analysis confirmed that the BP mRNAs are expressed from the late 4th instar to the adult stage with exception of prepupae and newly ecdysed pupae. While the BP1 transcript was prevalent in the larval stage, the BP2 transcript was abundant in the whole body only after the pupal stage in P. rapae. Both BPs were detected in a stage-specific pattern in the epidermis, testis, hindgut, wing, brain, and egg, with a lesser amount in the fat body. Two-dimensional gel electrophoresis and Western blot analyses revealed that BP1 was dominant in tissues from larvae, BP2 was dominant in tissues from pupal stages, and both BPs appeared in tissues from the adult stage, though BP2 was predominant.

Amino Acid Sequence↗
PubMed1987

Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor.

A family of peptides with broad-spectrum antimicrobial activity has been isolated from the skin of the African clawed frog Xenopus laevis. It consists of two closely related peptides that are each 23 amino acids and differ by two substitutions. These peptides are water soluble, nonhemolytic at their effective antimicrobial concentrations, and potentially amphiphilic. At low concentrations they inhibit growth of numerous species of bacteria and fungi and induce osmotic lysis of protozoa. The sequence of a partial cDNA of the precursor reveals that both peptides derive from a common larger protein. These peptides appear to represent a previously unrecognized class of vertebrate antimicrobial activities.

Amino Acid Sequence↗
PubMed2004

Comparison of genomic and cDNA sequences of guinea pig CYP2B18 and rat CYP2B2: absence of a phenobarbital-responsive enhancer module in the upstream region of the CYP2B18 gene.

Potential mechanisms were investigated whereby CYP2B18, a cytochrome P450 gene exhibiting high constitutive expression but only low levels of phenobarbital-inducibility in the guinea pig liver, may be differentially regulated versus the highly inducible rat CYP2B2 gene. To comparatively assess potential regulatory sequences associated with CYP2B18, a guinea pig genomic library was screened enabling isolation of the CYP2B18 gene. The genomic screening process resulted in the identification of at least four closely-related CYP2B18 genes, designated here as CYP2B18A-D. Of these isolates, CYP2B18A exhibited sequence identical to that of the CYP2B18 cDNA. Further, the deduced amino acid sequence of the CYP2B18 cDNA was identical to that of N-terminal and internally-derived peptide sequences obtained in this investigation from CYP2B18 protein isolated from guinea pig liver. Genomic structural sequences were derived for CYP2B18A, together with the respective 5'-upstream and intronic regions of the gene. Comparison of the CYP2B18A and CYP2B2 gene sequences revealed the lack of repetitive LINE gene sequences in CYP2B18A, putative silencing elements that effect neighboring genes, although these sequences were present in both 5'-upstream and 3'-downstream regions of CYP2B2. We determined that the phenobarbital-responsive enhancer module was absent from the 5'-upstream region as well as the intronic regions of CYP2B18A gene. We hypothesize that the compromised phenobarbital inducibility of CYP2B18A stems from its lack of a functional phenobarbital responsive enhancer module.

Amino Acid Sequence↗