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PubMed2005

Identification and cloning of caprine uterine serpin.

The uterine serpins have been described in sheep, cattle, and pigs as a highly diverged group of the large superfamily of serpin proteins that typically function as serine proteinase inhibitors. Here, the range of species that possess and express a uterine serpin gene is extended to the goat. Sequencing of cDNA amplified from total RNA from a pregnant goat at day 25 of pregnancy resulted in a 1,292 bp full-length consensus cDNA sequence for caprine uterine serpin (CaUS). The predicted amino acid sequence of the caprine precursor showed 96%, 82%, 55%, and 56% identity to OvUS, BoUS, PoUS1, and PoUS2, respectively. The signal peptide extends from amino acids 1 to 25, resulting in a secreted protein of 404 amino acids and 46,227 Mr (excluding carbohydrate). Both the goat and sheep uterine serpins have a nine amino acid insert in the Helix I region that is not found in bovine or porcine uterine serpins. A total of 13 amino acids in CaUS are different than those for the nearest homologue, ovine uterine serpin. One of these is in the site of cleavage of the signal sequence, where a single nucleotide substitution (G --> C) changed the cysteine for the sheep, bovine, and porcine genes to a serine. In addition, the amino acid at the putative P1-P1' site (the scissile bond for antiproteinase activity) is a valine for CaUS, BoUS, PoUS1, and PoUS2 versus an alanine for OvUS. The hinge region of all five of the uterine serpins (P17-P9) is distinct from the consensus pattern for inhibitory sequences and it is unlikely, therefore, that the uterine serpins possess prototypical proteinase inhibitory activity. The goat uterine serpin was immunolocalized to the glandular epithelium of the endometrium from a pregnant nanny at day 25 of pregnancy. There was also immunoreactive product in scattered luminal epithelial cells. No immunoreaction product was detected in endometrium from a nanny at day 5 of the estrous cycle. Western blotting of uterine fluid collected from the pregnant uterine horn of a unilaterally-pregnant goat revealed the presence of a protein band at Mr approximately 56,000 that reacted with monoclonal antibody to OvUS. In conclusion, the range of species in which uterine serpins are present and expressed in the uterus includes the goat in addition to the previously described sheep, cow, and pig. In all of these species, the uterine serpin is derived primarily from glandular epithelium, is secreted into the uterine lumen, and contains sequence characteristics suggesting it is not an inhibitory serpin.

Amino Acid Sequence↗
PubMed1996

Identification of a dual leucine zipper kinase involved in rat fracture repair.

Complete cDNA sequence was obtained for a gene found to be expressed at a high level during fracture healing in the rat. Comparison of the amino acid sequence (deduced from the cDNA sequence) with known sequences showed 99% identity with mouse Dual Leucine Zipper Kinase (mDLK) and 96% identity with human Zipper Protein Kinase (hZPK). Relative quantitation by RT-PCR indicates abundant expression of this gene in cultured chondrocytes, growth plate and brain tissue. Further, regulated expression of this gene has been observed during fracture healing in rat femur fracture model; maximum gene expression in this model appears to coincide with chondrogenesis and bone formation. Since DLK has been shown to function in the mitogen activated signaling (MAP) pathway, these results provide the first evidence for the involvement of this signaling pathway in fracture healing.

Amino Acid Sequence↗
PubMed1999

Studies on the GH/SL gene family: cloning of African lungfish (Protopterus annectens) growth hormone and somatolactin and toad (Bufo marinus) growth hormone.

The lungfishes (lobe-finned fish) occupy a unique position in vertebrate phylogeny, being regarded as the closest extant relatives to the tetrapods. The putative pituitary hormone somatolactin (SL) has hitherto been found only in teleost fishes, and the presence of this protein in tetrapods or lobe-finned fishes has not been ascertained. It was therefore of interest to determine the structure of SL in the African lungfish (Protopterus annectens), as this information would be useful for designing probes to facilitate the detection of SL genes in amphibians and other tetrapods. The structural relationships between SL, growth hormone (GH), and prolactin (PRL) strongly suggest that these proteins evolved from a common ancestor. To obtain a more complete picture of the evolution of these hormones in lungfish, African lungfish GH has been cloned and sequenced. The cDNA sequence of a toad (Bufo marinus) GH was determined to facilitate maximum parsimony analysis of GH sequences. Cladistic analysis confirmed that lungfish and amphibian GH sequences form a clade distinct from the GH sequences of ray-finned fishes. A distance matrix analysis of SL sequences indicated that lungfish SL had the lowest primary sequence identity with goldfish SL (47%) and the highest with flounder SL (66%). The detection of SL in a lungfish indicates that the gene duplication within the SL/GH/PRL family, which gave rise to SL, must have occurred in a common ancestor of the ray-finned fishes (Actinopterygii) and the lungfishes (Sarcopterygii) and tetrapods.

Amino Acid Sequence↗
PubMed1994

Complete coding sequence, deduced primary structure, chromosomal localization, and structural analysis of murine aggrecan.

We have isolated and sequenced overlapping cDNA clones encoding the entire core protein of aggrecan (the large aggregating chondroitin sulfate/keratan sulfate proteoglycan of cartilage) from three chondrocyte cDNA libraries of BALB/c mice and localized the aggrecan gene in mouse chromosome 7. We determined 7386 bp of the cDNA sequence, including 132 and 854 nucleotides of 5' and 3' untranslated regions, respectively. The core protein precursor is 2132 amino acids long (M(r) 222,008), including a 19-residue secretory signal peptide. The overall amino acid sequence of the mouse aggrecan shows 91.6% identity to rat and 72.5% to human aggrecan. Comparison of the amino acid sequences of various domains and subdomain structures of mouse aggrecan to known sequences of other species and related proteins (versican, neurocan, link protein, and lymphocyte homing receptor CD44) revealed high levels of identity of the G1, G2, and G3 globular domains and relatively less conserved structures in the interglobular and glycosaminoglycan-attachment regions. Epidermal growth factor (EGF)-like module was detected in only a minor fraction of aggrecan clones, while the complement regulatory protein (CRP)-like domain was regularly expressed in all samples.

Aggrecans↗
PubMed2002

Use of a common promoter by two juxtaposed and intronless mouse early embryonic genes, Rnf33 and Rnf35: implications in zygotic gene expression.

Rnf33 and Rnf35 are mouse RING finger protein genes that are transcribed temporally in the preimplantation mouse embryo, predominantly at the two-cell embryonic stage. The genes are juxtaposed in a 20-kb genomic region and are both intronless except for a single intron in the 5' untranslated region (5'-UTR). Based on analysis of the Rnf33/35 genomic sequence and cDNA sequences derived by in silico mining, we found that the Rnf33 and Rnf35 mRNAs are apparently transcribed from the same putative promoter and may be products of alternative splicing of the same pre-mRNA generated through differential 3' cleavage and polyadenylation. We also detected a second variant of Rnf35 in two-cell embryo generated through a second splicing event using an unconventional 5' splice junction. Our observations on the mode of transcription of Rnf33 and Rnf35 are consistent with the hypothesis that transcription of zygotic genes is promiscuous, and that the solo 5'-UTR intron may serve to facilitate efficient translation.

Animals↗
PubMed1993

Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana.

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.

2-Acetolactate Mutase↗
PubMed1992

The identification of leaf thionin as one of the main jasmonate-induced proteins of barley (Hordeum vulgare).

Jasmonic acid (JA) and its methyl ester (JA-Me) are able to introduce the accumulation of several specific polypeptides in cut leaf segments of barley. Two of the most prominent JA-induced proteins of M(r) 15,000 and 23,000 have been characterized by isolating and sequencing complete cDNA sequences. While the sequence of the M(r) 23,000 polypeptide shows no similarity to published sequences, the sequence of the M(r) 15,000 polypeptide corresponds to the higher-molecular-weight precursor of a leaf thionin previously characterized. Transcripts for the M(r) 23,000 and M(r) 15,000 polypeptides accumulate in leaf segments shortly after the beginning of JA treatment. JA and JA-Me induce the appearance of the two proteins not only in leaf segments but also in intact barley seedlings. However, in seedlings the accumulation of JA-induced proteins occurs much more slowly and requires high concentrations of volatile JA-Me. Thus, in barley it seems unlikely that volatile JA-Me is involved in the interaction between different members of this species, as has been proposed recently for tomato seedlings.

Amino Acid Sequence↗
PubMed1995

Fundulus heteroclitus vitellogenin: the deduced primary structure of a piscine precursor to noncrystalline, liquid-phase yolk protein.

We have cloned and sequenced a cDNA encoding a vitellogenin (Vtg) from the mummichog, Fundulus heteroclitus, an estuarine teleost. We constructed a liver cDNA library against RNA from estrogen-treated male mummichogs. Five overlapping cDNA clones totalling 5,197 bp were isolated through a combination of degenerate oligonucleotide probing of the library and PCR. The cDNA sequence contains a 5,112 bp open reading frame. The predicted primary structure of the deduced 1,704-amino-acid protein is 30-40% identical to other documented chordate Vtgs, establishing this Vtg as a member of the ancient Vtg gene family. Of the previously reported chordate Vtg sequences (Xenopus laevis, Gallus domesticus, Ichthyomyzon unicuspis, and Acipenser transmontanus), all four act as precursor proteins to a yolk which is eventually rendered insoluble under physiological conditions, either as crystalline platelets or as noncrystalline granules. The yolk of F. heteroclitus, on the other hand, remains in a soluble state throughout oocyte growth. The putative F. heteroclitus Vtg contains a polyserine region with a relative serine composition that is 10-20% higher than that observed for the other Vtgs. The trinucleotide repeats encoding the characteristic polyserine tracts of the phosvitin region follow a previously reported trend: TCX codons on the 5' end and AGY codons toward the 3' end. Whether the difference in Vtg primary structure between F. heteroclitus and that of other chordates is responsible for the differences in yolk structure remains to be elucidated. As the first complete teleost Vtg to be reported, these data will aid in designing nucleotide and immunological probes for detecting Vtg as a reproductive status indicator in F. heteroclitus and other piscine species.

Amino Acid Sequence↗
PubMedDate not supplied

Detection of seven point mutations in the porphobilinogen deaminase gene in patients with acute intermittent porphyria, by direct sequencing of in vitro amplified cDNA.

Direct cDNA sequencing has been performed on asymmetrically amplified transcripts from the human porphobilinogen deaminase gene. Lymphocytes from 30 patients with acute intermittent porphyria were the source of mRNA; of the seven separate point mutations detected, three were silent, whereas four resulted in amino acid changes. Three of these changes involved highly conserved amino acids, and the remaining one a conserved charge. One of these mutations was predicted to cause structural alterations in the protein product. The application of this method to affected families allows the direct identification of these heterogeneous mutations, thus permitting the unequivocal detection of carriers.

Base Sequence↗
PubMed2006

cDNA clone, fusion expression and purification of the novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E. coli.

Powdery mildew, caused by Uncinula necator Burr, is one of the most seriously damaging diseases of grapevine all over the world. To gain the novel gene and investigate the resistance mechanism in Chinese Wild Vitis pseudoreticulata clone Baihe-35-1, mRNA differential display was employed to study the differential expression of the resistant gene to the disease of it when inoculated by Uncinula necator under natural field conditions, 5' RACE and 3' RACE have been used to clone the whole cDNA sequences of VpAPX, the novel gene related to Ascorbate Peroxidase which involved in resistant to the disease, is composed of specific sequence 1077 bp and has an open reading frame of 750 bp coding for 250 amino acid residues with a molecular weight of 27.566 kDa. The VpAPX gene was obtained by polymerase chain reaction (PCR) with the special primers synthesized according to the sequences of cDNA, and further cloned it into the pGEM-T easy vector. The cloned VpAPX gene was cut out again with two restriction enzymes and was inserted into the prokaryotic expression vector pGEX-4T-1, then transferred into E. coli BL21. As result, GST-VpAPX fusion protein was successfully expressed by induction of IPTG and purified by GST affinity resin. After injecting rabbit, the polyclonal antibodies were produced. Western blot analyses showed that the antibody reacted specifically to GST-VpAPX fusion protein and the titer for this antibody is 10(5). This research made the foundation to transform the VpAPX gene into grape plants for follow research in processing.

Ascorbate Peroxidases↗
PubMed1989

A distinct form of tau is selectively incorporated into Alzheimer's paired helical filaments.

Tau, a microtubule-associated phosphoprotein, was identified as a definite component of paired helical filaments which progressively accumulate in Alzheimer's disease brain. To learn more about tau in the aged brain, we have isolated and sequenced a cDNA clone encoding tau from a cDNA library of an aged human brain. The cloned cDNA sequence included a new insert of 93 nucleotides, which added a fourth repeat to the three-repeat type of tau already reported. Perhaps, this four-repeat type of tau is predominant in normal aged brain. In contrast, the sequence analysis of paired helical filaments showed that the integrated tau is of three-repeat type. This indicates that a distinct form of tau is selectively incorporated into paired helical filaments.

Aged↗
PubMed1994

Isolation, biochemical characterization and N-terminal sequence of rolipram-sensitive cAMP phosphodiesterase from human mononuclear leukocytes.

A cyclic AMP specific phosphodiesterase (type IV) was purified 450,000-fold from human peripheral blood mononuclear cells through a sequence of chromatographic steps involving anion exchange, affinity chromatography on a matrix coupled to a derivative of the type IV inhibitor rolipram, and gel filtration. The enzyme showed apparent molecular masses of 70 kDa on gel filtration and 35 kDa on denaturing or native PAGE, indicating a possible dimerization or cleavage under certain conditions. The isoelectric point was 4.6. Kinetic parameters were Km = 2.2 microM, Ki = 1.2 microM (rolipram) and vmax = 80 mumol/min per mg protein. The most probable N-terminal sequence was determined as SLTNTNIPRF, 80% identical to part of the deduced amino acid sequence from cDNA sequences of PDE IVA and PDE IVD.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin.

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.

Amino Acid Sequence↗
PubMed1986

Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin.

We describe the isolation, characterization, and sequence of cDNA clones encoding one subunit of the complex of membrane glycoproteins that forms part of the transmembrane connection between the extracellular matrix and the cytoskeleton. The cDNA sequence encodes a polypeptide of 89 kd that has features strongly suggesting the presence of a large N-terminal extracellular domain, a single transmembrane segment, and a small C-terminal cytoplasmic domain. The extracellular domain contains a threefold repeat of a novel 40 residue cysteine-rich segment, and the cytoplasmic domain contains a tyrosine residue that is a potential site for phosphorylation by tyrosine kinases. We propose the name integrin for this protein complex to denote its role as an integral membrane complex involved in the transmembrane association between the extracellular matrix and the cytoskeleton.

Actins↗
PubMed1992

Sequence characterization of a sheep cDNA for antithrombin III.

We determined the cDNA sequence of the mRNA for antithrombin III (AT III) from sheep liver. It encodes a protein of 465 amino acids, including a signal peptide of 32 amino acids. The amino acid sequence of the mature protein shows a sequence identity of 89.1%, 95.6% and 85.0% to the human, bovine and rabbit equivalents, respectively. Cysteine residues involved in disulfide bonds as well as potential glycosylation sites are conserved between the four species. In contrast, the amino acid sequence of the signal peptide shows a smaller identity, i.e., 68.7% and 56.3% compared to the human and rabbit preprotein, respectively.

Amino Acid Sequence↗
PubMed1993

Cloning and sequence analysis of a murine cDNA encoding glutamate decarboxylase (GAD65).

We report the cloning and cDNA sequence of murine GAD65. Murine GAD65 is comprised of 585 amino acids and shares a high degree of homology with human and rat GAD65, with most divergences occurring near their amino-termini. The murine GAD65 sequence will allow evaluation of the role of this gene in murine neurogenetic and autoimmune diseases.

Amino Acid Sequence↗
PubMed2005

Molecular cloning and comparative analysis of fibrinogen-related proteins from the soft tick Ornithodoros moubata and the hard tick Ixodes ricinus.

Among disease-vectors, the evolution of the tick innate immune system is still lagging when compared to insects. Such an investigation, which was initiated, by first cloning and sequencing lectins associated in the innate immunity of invertebrates and having fibrinogen related domains, helped in the sequencing of cDNA encoding for OMFREP from the soft tick, Ornithodoros moubata. Also obtained were Ixoderin A and Ixoderin B cDNA sequences from the hard tick Ixodes ricinus. Tissue-specific expression of OMFREP showed that it was present primarily in the hemocytes and salivary glands. Ixoderin A besides sharing a similar expression profile was also expressed in the midgut. Both showed significantly high homology to the lectin Dorin M, from O. moubata. Further, phylogenetic comparisons between these molecules of the soft and hard ticks showed their relatedness to Tachylectins 5A and 5B, involved in the innate immunity of Tachypleus tridentatus and ficolins from both vertebrates and invertebrates. Ixoderin B showing tissue-specific expression only in the salivary glands and the sequence displaying certain motif differences in homology point towards a possible function different from the other two molecules. This is the first report of lectin-like sequences, with a fibrinogen-domain, from the hard tick I. ricinus and a preliminary phylogenetic study of these tick sequences with related fibrinogen-domain containing sequences highlights a possible role for them in the innate immunity of the ticks.

Amino Acid Sequence↗
PubMed2006

Protein and genomic organisation of vertebrate MyoR and Capsulin genes and their expression during avian development.

The related bHLH transcription factors MyoR and Capsulin control craniofacial myogenesis and the development of a number of mesoderm-derived organs in the mouse. However, their molecular function as regulators of differentiation processes is conversely debated. One approach to clarify the roles of these genes is to comparatively analyse their biological and molecular function in various vertebrate models. For this, a prerequisite is the determination of their similarity and their expression patterns. Here we show that vertebrate MyoR and Capsulin are paralogous genes with a high level of conservation regarding their protein sequence, their cDNA sequence and their chromosomal organisation. In the chick, both genes are co-expressed in the developing branchiomeric muscles, the anterior heart field and the splanchnopleura lining the foregut. However, both genes show unique expression domains in trunk skeletal muscle precursors, in the lateral and intermediate mesoderm.

Animals↗