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PubMed1982

Molecular cloning of a cDNA sequence encoding a trophectoderm-specific marker during mouse blastocyst formation.

A cDNA clone has been isolated from a trophoblastoma cDNA library. The mRNA complementary to this sequence directs the in vitro synthesis of proteins. The two-dimensional electrophoretic pattern of migration of these proteins is superposable to that of the trophoblastoma intermediate filament proteins recognized by monoclonal antibody (mAb) TROMA-1. This mAb had previously been shown to label trophectoderm cells but not inner cell mass cells. With a sensitive binding assay (ultrasensitive enzymatic radioimmunoassay), these in vitro synthesized proteins were recognized by mAb TROMA-1. The proteins are immunoprecipitated by an antiserum directed against trophoblastoma intermediate filament proteins and by a serum directed against a major cytoskeletal protein found in murine extraembryonic endodermal cell lines (Endo-A) [Oshima R. G. (1981) J. Biol. Chem. 256, 8124-8133]. The cDNA sequence detects specific mRNA(s) migrating with 18S ribosomal RNA in trophoblastoma but not in embryonal carcinoma cells.

Animals↗
PubMed1997

Molecular characterisation of a cDNA sequence encoding the backbone of a style-specific 120 kDa glycoprotein which has features of both extensins and arabinogalactan proteins.

Nicotiana alata has a style-specific hydroxyproline-rich glycoprotein (the 120 kDa glycoprotein) which has properties of both extensins and AGPs [19, 20]. The 120 kDa glycoprotein is a soluble component in the extracellular matrix of the transmitting tract of styles where it accounts for ca. 9% of the total buffer-soluble protein. Here we describe the molecular cloning of a cDNA representing the gene NaPRP5 which encodes the backbone of the 120 kDa glycoprotein. Expression of mRNA is restricted to styles, consistent with observations on the distribution of the 120 kDa glycoprotein. Levels of accumulation of the transcript encoding the 120 kDa protein backbone are not altered significantly by pollination with either compatible or incompatible pollen. The protein backbone of the 120 kDa glycoprotein, as predicted by the cDNA sequence, is composed of three distinct domains. The sequence of these domains, together with linkage analysis of the carbohydrate component of the 120 kDa glycoprotein, allows predictions of the likely distribution of substituent glycosyl chains along the protein backbone. The similarity of the C-terminal domains of the 120 kDa glycoprotein and GaRSGP, the galactose-rich style glycoprotein of N. alata, is consistent with the two molecules sharing a common antigenic domain in their backbones [31]. The sharing of domains between distinct hydroxyproline-rich glycoproteins suggests that identification of a glycoprotein of this class solely by its protein or carbohydrate epitope is not valid.

Amino Acid Sequence↗
PubMed1992

Evolutionary implications of the cDNA sequence of the single lactate dehydrogenase of a lamprey.

All vertebrates other than lampreys exhibit multiple loci encoding lactate dehydrogenase +ADL-LDH; (S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27+BD. Of these loci, Ldh-A is expressed predominantly in muscle, Ldh-B is expressed predominantly in heart, and Ldh-C (where present) exhibits different tissue-restricted patterns of expression depending on the taxon. To examine the relationship of the single LDH of lampreys to other vertebrate LDHs, we have determined the cDNA sequence of the LDH of the sea lamprey Petromyzon marinus and compared it to previously published sequences from bacteria, plants, and vertebrates. The lamprey sequence exhibits a mixture of features of both LDH-A and LDH-B at the amino acid level that may account for its intermediate kinetic properties. Both distance and maximum parsimony analyses strongly reject a relationship of lamprey LDH with mammalian LDH-C but do not significantly distinguish among remaining alternative phylogenetic hypotheses. Evolutionary parsimony analyses suggest that the lamprey LDH is related to Ldh-A and that the single locus condition has arisen as a result of the loss of Ldh-B (prior to the appearance of Ldh-C). The collection of LDH sequences for further studies of the evolution of the vertebrate LDH gene family will be facilitated by the PCR approach that we have used to obtain the lamprey sequence.

Amino Acid Sequence↗
PubMed2000

Full-length cDNA sequence and genomic organization of human NKX3A - alternative forms and regulation by both androgens and estrogens.

NKX3A (NKX3.1) is a recently identified androgen-regulated gene that is largely specific to prostate for expression and likely to code for a homeobox protein. Here we report the full-length mRNA and genomic organization of human NKX3.1. There are at least five different splice variants of NKX3.1 mRNA that result in different open reading frames (ORFs). There is extensive similarity between the human and the mouse NKX3.1 cDNA sequences outside of the ORFs (greater than 60% overall identity), which may be involved in modulating NKX3.1 expression. In addition to its androgen regulation in the prostate cancer cell line LNCaP, we show that NKX3.1 expression is androgen-dependent in the CWR22 prostate cancer xenograft model. Interestingly, NKX3.1 is highly expressed in the androgen-independent derivative CWR22R in the absence of androgens, indicating that it may be deregulated in advanced prostate cancer. Using a Green Fluorescent Protein fusion construct, we show that NKX3.1 is a nuclear protein consistent with its proposed function as a homeobox transcription factor. Furthermore, in addition to androgens, NKX3.1 expression is up-regulated by 17beta-estradiol, but not by progesterone, dexamethasone, or 3,5,3'-triiodothyronine in LNCaP cells. Regulation of NKX3.1 by androgens and 17beta-estradiol in prostate cancer cells and its deregulation in androgen-independent prostate cancer suggest that it may have important regulatory roles during prostate cancer progression.

Alternative Splicing↗
PubMed2000

Purification, characterization, and cDNA sequencing of cytosolic phospholipase A(2) from equine neutrophils.

It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence demonstrated 95% identity with human and mouse cPLA(2), as well as 83 and 73% identity with chicken and zebra fish cPLA(2) protein, respectively. The equine cPLA(2) possessed some properties that distinguished the equine enzyme from the human enzyme. First, the enzyme activity of the equine cPLA(2) was differently influenced by cations as compared with the human cPLA(2). Second, the equine neutrophil cPLA(2) migrated as an approximately 105-kDa protein, in comparison with human cPLA(2) which migrated as a 110-kDa protein. A difference between equine neutrophils and eosinophils in the degree of phosphorylation of the cPLA(2) protein was observed. Thus, the cPLA(2) protein from eosinophils was constitutively phosphorylated, while the cPLA(2) protein from neutrophils was unphosphorylated. In summary, these results demonstrate that equine neutrophils indeed express an active cPLA(2) protein but that there is a difference in the degree of phosphorylation of the cPLA(2) protein between equine neutrophils and eosinophils. This difference might explain the difference between the two cell types in the capacity to produce leukotrienes from endogenous substrate.

Amino Acid Sequence↗
PubMed1999

Rapid evolution of fertilization selectivity and lysin cDNA sequences in teguline gastropods.

Proteins mediating intercellular recognition face opposing selective forces as they evolve: purifying selection to maintain function, and diversifying selection to alter specificity. Lysin is a 16-kDa protein which enables sperm of free-spawning marine snails to make a hole in the vitelline layer (VE) surrounding conspecific eggs. Previous work on abalone (Haliotis spp.) has shown that positive selection promotes rapid interspecific divergence of lysin. Here, we present data on the specificity of VE dissolution by four species of teguline gastropods, along with lysin cDNA sequences. The teguline and abalone lineages diverged over 250 MYA. As in abalone, VE dissolution by lysin in tegulines is species-selective, and positive selection promotes rapid interspecific divergence over the entire mature protein. Nonsynonymous substitution rates, calculated using a mtCOI molecular clock calibrated by two Tegula species separated by the Isthmus of Panama, are high (> 25 substitutions per site per 10(9) years). However, the extensive replacements in teguline lysins are overwhelmingly conservative with respect to type, charge, and polarity of residues. Predictions of secondary structure suggest that the size and position of alpha-helices are also conserved, even through pairwise amino acid identities between Haliotis rufescens and the different tegulines are less than 15%.

Amino Acid Sequence↗
PubMed1988

Drosophila basement membrane procollagen alpha 1(IV). II. Complete cDNA sequence, genomic structure, and general implications for supramolecular assemblies.

A Drosophila melanogaster gene for a basement membrane procollagen chain was recently identified from the sequence homology of the carboxyl (NC1) end of the polypeptide that it encodes with the corresponding domain of human and murine collagens IV (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). This gene is at chromosome location 25C. Here we report the complete 6-kilobase cDNA sequence coding for a chain of 1775 amino acids, as well as the genomic structure. The gene is composed of nine relatively large exons separated by eight relatively small introns. This organization is different from the multiple small exons separated by large introns reported for mouse and human type IV collagens (Kurkinen, M., Bernard, M. P., Barlow, D. P., and Chow, L. T. (1985) Nature 317, 177-179. Sakurai, Y., Sullivan, M., and Yamada, Y. (1986) J. Biol. Chem. 261, 6654-6657. Soininen, R., Tikka, L., Chow, L., Pihlajaniemi, T., Kurkinen, M., Prockop, D. J., Boyd, C. D., and Tryggvason, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1568-1572). Drosophila and human alpha 1(IV) procollagen chains share not only polypeptide domains near their amino and carboxyl ends for making specialized, intermolecular junctional complexes, but also 11 of 21 sites of imperfections of the collagen triple helix. However, neither the number nor the nature of the amino acids in these imperfections appear to have been conserved. These imperfections of the helical sequence may be important for the supramolecular assembly of basement membrane collagen. The 9 cysteine residues of the Drosophila collagen thread domain are arranged as several variations of a motif found in vertebrate collagens IV only near their amino ends, in their "7 S" junctional domains. The relative positions of these cysteine residues provide numerous opportunities for disulfide bonding between molecules in both parallel and antiparallel arrays. There is a pseudorepeat of one-third of the thread length, and there are numerous possibilities for disulfide-linked microfibrils and networks. We propose that collagen microfibrils, stabilized by disulfide segment junctions, are a versatile ancestral form from which specialized collagen fibers and networks arose.

Amino Acid Sequence↗
PubMed1991

The salmon gene encoding apolipoprotein A-I: cDNA sequence, tissue expression and evolution.

A cDNA encoding an apolipoprotein (Apo) has been isolated from the Atlantic salmon (Salmo salar) and sequenced. It encodes a peptide of 258 amino acids (aa), including a signal peptide of 18 aa, with 5'- and 3'-untranslated regions of the mRNA of 12 and 329 nucleotides, respectively. The protein has structural features in common with other Apo's of human and avian origin, including conserved sequences in the signal peptide and a series of internal repeats of 22 aa. The sequence has been identified as salmon Apo A-I (sApoA-I), and has 23% aa identity with human ApoA-I. Northern-blot analysis using the sApoA-I cDNA probe against total RNA prepared from several salmon tissues detects the expression of this gene in liver, intestine and muscle. A phylogenetic analysis reveals that the mammalian ApoA-I, ApoA-IV and Apo-E aa sequences are more closely related to each other than any of them are to sApoA-I. This suggests that the duplication events, from which A-I, A-IV and E arose, occurred after the divergence of the tetrapod and teleost ancestors.

Amino Acid Sequence↗
PubMed1991

A Xenopus laevis creatine kinase isozyme (CK-III/III) expressed preferentially in larval striated muscle: cDNA sequence, developmental expression and subcellular immunolocalization.

A cDNA containing the nearly complete coding sequence of CK-III subunit of X. laevis was isolated, sequenced and further identified by comparing the tissue distribution of CK-III/III isozyme with that of its messenger. Comparison of CK-III deduced amino acid sequence with other CK sequences published reveals its close homology to M-CK subunits. Results using both cDNA probes and monoclonal antibodies specific for CK-III subunits indicate that the appearance and the accumulation of CK-III occur in parallel with myoblast differentiation. Moreover, subcellular immuno-histolocalization shows that CK-III/III isozyme is especially concentrated on larval myofibres at the level of A-bands.

Amino Acid Sequence↗
PubMed1993

Cloning and cDNA sequence analysis of nephritogenic monoclonal antibodies derived from an MRL/lpr lupus mouse.

Production of IgG3 in MRL/Mp-lpr/lpr (MRL/lpr) lupus mice is one of the major factors to develop glomerulonephritis (GN) in these mice. To examine molecular characteristics of IgG3 responsible for GN in these mice, hybridoma clones producing IgG3 antibodies were prepared from one unmanipulated MRL/lpr mouse. Two clones, 2B11.3 and 7B6.8, were nephritogenic; that is, they caused severe glomerular lesions when injected to normal mice, moreover with a different histopathological manifestation. The 2B11.3 clone generated diffuse cell-proliferative lesions, while those induced by the 7B6.8 clone resembled wire loop lesions in human lupus nephritis. The cDNA sequence analysis of 7B6.8 antibody and the other IgG3 antibody, 1G3, non-nephritogenic, revealed that the C regions of the heavy and light kappa chains were completely the same between them. Furthermore, they were identical in deduced amino acid sequences to those from non-autoimmune BALB/c mice, indicating no allelic difference of Igh-8 between these two strains. The V regions of 2B11.3 and 7B6.8 antibodies were composed of different sets of VH, D, JH, Vk and Jk. Although both of the VH belonged to the J558 family, they seemed to use a different VH germline gene. These findings suggest that GN in MRL/lpr mice is generated by the expansion of clonally different B cells producing particular antibodies possibly with a different pathogenetic potency.

Amino Acid Sequence↗
PubMed1995

Variable region cDNA sequences and characterization of murine anti-human interferon gamma receptor monoclonal antibodies that inhibit receptor binding by interferon gamma.

Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.

Amino Acid Sequence↗
PubMed1999

Human eukaryotic initiation factor EIF2C1 gene: cDNA sequence, genomic organization, localization to chromosomal bands 1p34-p35, and expression.

We report the cloning and characterization of the human eukaryotic protein translation initiation factor EIF2C1 gene. The human EIF2C1 gene consists of 19 exons and 18 introns that span a region of almost 50 kb. It is located on the short arm of chromosome 1 in the region 1p34-p35. This genomic region is frequently lost in human cancers such as Wilms tumors, neuroblastoma, and carcinomas of the breast, liver, and colon. The human EIF2C1 gene is ubiquitously expressed at low to medium levels. Differential polyadenylation and splicing result in a complex transcriptional pattern. The cDNA sequence is 7478 bp long and contains an extremely large 3' untranslated region of 4799 bp with multiple, short repeated segments composed of mono-, tri-, or quattronucleotides interspersed throughout. The human EIF2C1 gene belongs to a multigene family in human. It is highly conserved during evolution, sharing about 90% identity with rabbit eIF2C and 70% identity with plant AGO1 at the amino acid level. These facts suggest that human EIF2C1 might play an important physiological role.

Adult↗
PubMed1992

Cloning and sequencing of a trophoblast-endothelial-activated lymphocyte surface protein: cDNA sequence and genomic structure.

We have previously described the distribution of a surface glycoprotein recognized by monoclonal antibody B721. We now report the molecular characterization of this molecule at the protein, cDNA, and genomic level. A 75-kDa glycoprotein can be immunoprecipitated from B721+ tissues. We have isolated a near full-length cDNA containing the complete coding region and a full-length genomic clone. We present evidence that this protein has similarity to several classes of nuclear transcription factors, particularly the myc family of proteins. The 721P protein was found to have a leucine zipper-like structure, a possible basic region immediately upstream from the leucine zipper, and a protein kinase A phosphorylation site. 721P protein is encoded by a gene distinct from any deposited in existing data bases, and displays several features associated with proteins involved in signal transduction and gene regulation.

Amino Acid Sequence↗
PubMed1991

Removal of 3' nontranslated cDNA sequence improves interferon gamma synthesis in Escherichia coli.

The cDNA coding for human interferon gamma contains approximately 600 basepairs not translated sequence with 4 adenine/thymidine (AT)-rich clusters at its 3'end. The influence of this sequence on the expression of recombinant human Interferon gamma in Escherichia coli was investigated. Removal of the non-translated sequence and especially of the AT-rich clusters yielded an increase in expression of interferon gamma from 10 to 40% of total protein. These results may also be important for the production of other recombinant human proteins with similar 3' sequences in their cDNA.

Bacterial Proteins↗
PubMed1998

cDNA sequence and chromosomal localization of mouse Dlgh3 gene adjacent to the BRCA1 tumor suppressor locus.

Membrane associated guanylate kinase homologues (MAGUKs) function in tumor suppression and receptor clustering pathways presumably by modulating signaling events at the interface of the membrane cytoskeleton. The p55 subclass of MAGUKs includes two novel cDNAs that were originally identified by virtue of their genomic location to human chromosome 17q12-21 where the BRCA1 tumor suppressor gene has been mapped. The predicted primary structure of the human MPP3 contains a single copy of the PDZ domain, an SH3 motif, and a carboxy-terminal guanylate kinase-like domain. Here we report the full-length coding cDNA sequence of the mouse homologue of MPP3. The translated amino acid sequence of murine Dlgh3 contains 568 amino acids that show 87% sequence identity with the human MPP3 protein. Northern blot analysis shows abundant expression of a approximately 3.0 kb transcript of Dlgh3 in mouse brain and skeletal muscle, and a relatively less abundant approximately 5.0 kb transcript in skeletal muscle, testis, kidney, and lung. Using an interspecific backcross panel, the Dlgh3 gene was mapped to a segment of mouse chromosome 11 that is conserved with human chromosome 17q12-21. The close proximity of murine Dlgh3 gene to the BRCA1 locus and the high conservation of the primary structure of human and murine proteins provide a framework for testing the role of Dlgh3 in cell proliferation pathways using the mouse as a model system.

Amino Acid Sequence↗
PubMed1994

cDNA sequence, gene sequence, and properties of murine pallidin (band 4.2), the protein implicated in the murine pallid mutation.

Band 4.2, which plays an important but poorly understood role in erythrocyte function and survival, is a major component of erythrocyte membranes. Recently, it has been shown that the gene for murine protein band 4.2 colocalizes on chromosome 2 with the murine pallid mutation, which affects the formation or function of intracellular storage granules in melanocytes and platelets and lysosomes in kidney. As a first step in identifying the mutation responsible for the pallid phenotype, we have sequenced the entire normal murine band 4.2 gene. Our results show that the gene for murine band 4.2 is approximately 22 kb in size, with 13 exons and 12 intervening introns. The organization of the mouse band 4.2 gene is identical to that of the human band 4.2 gene and similar to that of the genes for the transglutaminase enzymes, reiterating the membership of protein band 4.2 in the transglutaminase gene superfamily. We also present 3.5 kb of normal murine erythroid band 4.2 cDNA sequence containing an open reading frame of 2073 bp and coding for 691 amino acids. This is the same size as the human erythrocyte protein, with which the murine protein shares a 72% amino acid identity.

Amino Acid Sequence↗
PubMed1982

The cDNA sequence of a human epidermal keratin: divergence of sequence but conservation of structure among intermediate filament proteins.

We have determined the DNA sequence of a cloned cDNA that is complementary to the mRNA for the 50 kilodalton (kd) human epidermal keratin. This provides the first amino acid sequence for a cytoskeletal keratin. Comparison of this sequence with those of other keratins reveals an evolutionary relationship between the cytoskeletal and the microfibrillar keratins, but shows no homology to matrix or feather keratins. The 50 kd keratin shares 28%-30% homology with partial sequences of other intermediate filament proteins, which suggests that keratins may be the most distantly related members of this class of fibrous proteins. Our computer analyses predict that the 50 kd keratin contains two long alpha-helical domains separated by a cluster of helix-inhibitory residues in the middle of the protein. These findings indicate that despite major sequence divergence among intermediate filament proteins, they retain sequences compatible with secondary structural features that appear to be common to all of them.

Amino Acid Sequence↗
PubMed1997

Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I.

Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chID, bchH/chIH and bchI/chII encode proteins which are required for in vitro Mg-chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchI have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment. Protein-protein interaction among the three subunits CHL D, H and I was studied using the yeast two-hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X-Gal-containing agar plates. In vitro Mg2+ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg-chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.

Amino Acid Sequence↗