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At least 307 records · Page 17Linked to original sources

PubMed2002

Cloning and expression of a type IX-like collagen in tissues of the ascidian Ciona intestinalis.

Collagens are highly preserved proteins in invertebrates and vertebrates. To identify the collagens in urochordates, the total RNA extracted from the pharynx of the ascidian Ciona intestinalis was hybridized with a heterologous probe specific for the echinoderm Paracentrotus lividus fibrillar type I-like larval collagen. Using this probe, two main bands (i.e. 6 and 2.8 kb mRNA) were observed on Northern blot hybridization. The cDNA library prepared from poly(A)+RNA extracted from pharyngeal tissue was screened and a cDNA that specifies a type IX-like collagen was identified. This molecule presents a conceptual open reading frame for a protein containing 734 amino acids. In particular, we showed a 1 alpha chain type IX-like collagen characterized by three short triple-helical domains interspersed with four non-triple-helical sequences, with structural features of fibril-associated collagens with interrupted triple-helices (FACIT) collagens. Northern blot hybridizations indicate a 2.8 kb transcript size. Sequence comparison indicated homology (47.64%, 48.95%) between the type IX-like collagen of C. intestinalis and mouse and human type IX collagen. In situ hybridization of tunic and pharynx tissues shows the presence of transcripts in connective tissue cells.

Amino Acid Sequence↗

PubMed1996

Molecular cloning, expression and characterization of a recombinant glutathione S-transferase from Echinococcus multilocularis.

We report the identification and characterization of the first cestode glutathione S-transferase (GST) cDNA sequence. A fragment of an Echinococcus multilocularis glutathione S-transferase cDNA was isolated by the polymerase chain reaction. Subsequently, a Lambda zap cDNA library prepared from mRNA from protoscolices of E. multilocularis was screened with this PCR fragment. A complete cDNA clone was isolated and the nucleotide sequence determined. Analysis of the E. multilocularis GST-deduced amino acid sequence indicates that it is clearly related to the mammalian mu-class GSTs. The E. multilocularis GST cDNA was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. The 25.5-kDa enzyme subunit was purified to homogeneity using glutathione-sepharose chromatography. Gel filtration demonstrated that this GST is enzymatically active as a homodimer. The recombinant enzyme had conjugating activity with organic hydroperoxides and with members of the trans,trans-2,4 alkadienal and trans-2-alkenal series, which are secondary products of lipid peroxidation.

Amino Acid Sequence↗

PubMed2004

The potato P locus codes for flavonoid 3',5'-hydroxylase.

The potato P locus is required for the production of blue/purple anthocyanin pigments in any tissue of the potato plant such as tubers, flowers, or stems. We have previously reported, based on RFLP mapping in tomato, that the gene coding for the anthocyanin biosynthetic enzyme flavonoid 3',5'-hydroxylase (f3'5'h) maps to the same region of the tomato genome as P maps in potato. To further evaluate this association a Petunia f3'5'h gene was used to screen a potato cDNA library prepared from purple-colored flowers and stems. Six positively hybridizing cDNA clones were sequenced and all appeared to be derived from a single gene that shares 85% sequence identity at the amino acid level with Petunia f3'5'h. The potato gene cosegregated with purple tuber color in a diploid F1 sub-population of 37 purple and 25 red individuals and was found to be expressed in tuber skin only in the presence of the anthocyanin regulatory locus I. A potato f3'5'h cDNA clone was placed under the control of a doubled CaMV 35S promoter and introduced into the red-skinned cultivar 'Desiree'. Tuber and stem tissues that are colored red in Desiree were purple in nine of 17 independently transformed lines.

Amino Acid Sequence↗

PubMed1996

Phosphate transporters from the higher plant Arabidopsis thaliana.

Two cDNAs (AtPT1 and AtPT2) encoding plant phosphate transporters have been isolated from a library prepared with mRNA extracted from phosphate-starved Arabidopsis thaliana roots, The encoded polypeptides are 78% identical to each other and show high degree of amino acid sequence similarity with high-affinity phosphate transporters of Saccharomyces cerevisiae, Neurospora crassa, and the mycorrhizal fungus Glomus versiforme. The AtPT1 and AtPT2 polypeptides are integral membrane proteins predicted to contain 12 membrane-spanning domains separated into two groups of six by a large charged hydrophilic region. Upon expression, both AtPT1 and AtPT2 were able to complement the pho84 mutant phenotype of yeast strain NS219 lacking the high-affinity phosphate transport activity. AtPT1 and AtPT2 are representatives of two distinct, small gene families in A. thaliana. The transcripts of both genes are expressed in roots and are not detectable in leaves. The steady-state level of their mRNAs increases in response to phosphate starvation.

Amino Acid Sequence↗

PubMed1997

Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing.

We report the isolation and characterization of a third isoform of placenta growth factor (PlGF), generated by alternative splicing of its RNA transcript. This novel form of PlGF, PlGF-3, was cloned by the polymerase chain reaction technique using a human cDNA library prepared from the terminal placental tissue. PlGF-3 contains an in-frame insertion of 72-amino acids near the C-terminal portion of PlGF-1. Southern blot analysis revealed that a single gene encoded PlGF-2 and PlGF-3. Nucleic acid sequence analysis found the insertion of 216 nucleotides of PlGF-3 between exon 4 and exon 5 of the PlGF gene. Northern blot and tissue distribution studies discovered two mRNA species for PlGF-3, which were both uniquely expressed in the placenta. Transient expression of PlGF-3 cDNA in mammalian cells showed PlGF-3 was detected in the conditioned medium as both dimers and monomers. Unlike PlGF-2, PlGF-3 lacked heparin-binding affinity. Thus, alternative splicing of PlGF RNA produces at least three polypeptides with different secretion pattern, heparin-binding affinity and dimerization properties.

Alternative Splicing↗

PubMed1993

Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm.

The venom of honeybees, Apis mellifera, contains several biologically active peptides and two enzymes, one of which is a hyaluronidase. By using degenerate oligonucleotides derived from the amino-terminal sequence of this hyaluronidase reported by others, clones encoding the precursor for this enzyme could be isolated from a cDNA library prepared from venom glands of worker bees. The deduced amino acid sequence showed that bee venom hyaluronidase is a polypeptide composed of 349 amino acids containing four cysteines and three potential sites for N-glycosylation. The sequence of the precursor also indicated that the conversion of the pro-enzyme to the end product must involve cleavage of a Thr-Pro bond, a most unusual processing reaction. The mRNA encoding hyaluronidase could also be detected in testes from drones. Expression of the cloned cDNA in Escherichia coli yielded a 41-kDa polypeptide that had hyaluronidase activity. Interestingly, the hyaluronidase from bee venom glands exhibited significant homology to PH-20, a membrane protein of guinea pig sperm involved in sperm-egg adhesion. These structural data support the long-held view that hyaluronidases play a role in fertilization.

Acrosome↗

PubMed1990

Molecular cloning, nucleotide sequence and expression in Escherichia coli of the beta-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8.

The beta-cyclodextrin glycosyltransferase (beta-CGTase) gene was isolated from a lambda-library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant beta-CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme.

Amino Acid Sequence↗

PubMed1991

Cloning and characterization of the ribosomal RNA gene repeat from Ostertagia ostertagi.

Clones of the rRNA genes of Ostertagia ostertagi were selected from a library prepared from the genomic DNA of adult worms of strain LA-2. A 13.4-kb insert in a clone, lambda OOR78, is comprised of one complete 7.5-kb rDNA unit and portions of two adjacent units. The rDNA unit is directly repeated in a head-to-tail fashion and represents approximately 0.9% of the total genomic DNA. This repeating unit appears to be the only long tandemly repeated sequence in the genome. Restriction enzyme recognition sites in the rDNAs of four strains of O. ostertagi were fully conserved with the exception of one PstI site present in the large rRNA gene which was absent from a proportion of the genes of the LA-2 strain. The rDNA of O. ostertagi is more similar to that of Caenorhabditis elegans in unit length and arrangement than to parasitic helminths previously examined.

Animals↗

PubMed2005

BnNHL18A shows a localization change by stress-inducing chemical treatments.

The two genes, named BnNHL18A and BnNHL18B, showing sequence homology with Arabidopsis NDR1/HIN1-like (NHL) genes, were isolated from cDNA library prepared with oilseed rape (Brassica napus) seedlings treated with NaCl. The transcript level of BnNHL18A was increased by sodium chloride, ethephon, hydrogen peroxide, methyl jasmonate, or salicylic acid treatment. The coding regions of BnNHL18A and BnNHL18B contain a sarcolipin (SLN)-like sequence. Analysis of the localization of smGFP fusion proteins showed that BnNHL18A is mainly localized to endoplasmic reticulum (ER). This result suggests that the SLN-like sequence plays a role in retaining proteins in ER membrane in plants. In response to NaCl, hydrogen peroxide, ethephon, and salicylic acid treatments, the protein localization of BnNHL18A was changed. Our findings suggest a common function of BnNHL18A in biotic and abiotic stresses, and demonstrate the presence of the shared mechanism of protein translocalization between the responses to plant pathogen and to osmotic stress.

Acetates↗

PubMed1997

Human autoimmune anti-proteinase 3 scFv from a phage display library.

This is the first study describing recombinant human antibody fragments directed to the autoantigen proteinase 3 (PR3) from an immune B cell source. Detection of these autoantibodies has proven valid for the diagnosis and monitoring of Wegener's granulomatosis. The described antibody fragment (scFv) was isolated from a phage display library prepared from the IgG-positive splenic lymphocytes of a patient with systemic autoimmunity. The cloning strategy was designed to maintain the diversity of the antibody variable gene repertoire, and sequencing of several variable genes demonstrated that all major heavy and light chain families were represented. We found an over-representation of particular heavy chain variable domains in splenic lymphocytes which differ from the ones frequently found in peripheral blood lymphocytes. It was possible to obtain specific scFv to PR3 after a single round of selection and the binding could be inhibited by the patients' sera. Although the antibody fragments in the splenic repertoire were found to be highly mutated, it was interesting to find that the selected scFv showed only limited somatic mutation. Furthermore, we could demonstrate that the removal of the mutations had no effect on binding specificity.

Amino Acid Sequence↗

PubMed1995

iPABP, an inducible poly(A)-binding protein detected in activated human T cells.

The poly(A)-binding protein (PABP) binds to the poly(A) tail present at the 3' ends of most eukaryotic mRNAs. PABP is thought to play a role in both translation and mRNA stability. Here we describe the molecular cloning and characterization of an inducible PABP, iPABP, from a cDNA library prepared from activated T cells. iPABP shows 79% sequence identity to PABP at the amino acid level. The RNA binding domains of iPABP and PABP are nearly identical, while their C termini are more divergent. Like PABP, iPABP is primarily localized to the cytoplasm. iPABP is expressed at low levels in resting normal human T cells; following T-cell activation, however, iPABP mRNA levels are rapidly up-regulated. In contrast, PABP is constitutively expressed in both resting and activated T cells. iPABP mRNA was also expressed at much higher levels than PABP mRNA in heart and skeletal muscle tissue. These data suggest that the regulation of cytoplasmic poly(A)-binding activity is more complex than previously believed. In most tissues, poly(A)-binding activity is likely to be the result of the combined effects of constitutively expressed PABP and iPABP, whose expression is subject to more complex regulation.

Amino Acid Sequence↗

PubMed2004

Cloning and expression of an acidic platelet aggregation inhibitor phospholipase A2 cDNA from Bothrops jararacussu venom gland.

The phospholipase A2 (PLA2, E.C. 3.1.1.4) superfamily is defined by enzymes that catalyze the hydrolysis of the sn-2 bond of phosphoglycerides. Most PLA2s from the venom of Bothrops species are basic proteins, which have been well characterized both structurally and functionally, however, little is known about acidic PLA2s from this venom. Nevertheless, it has been demonstrated that they are non-toxic, with high catalytic and hypotensive activities and show the ability to inhibit platelet aggregation. To further understand the function of these proteins, we have isolated a cDNA that encodes an acidic PLA2 from a cDNA library prepared from the poly(A)+ RNA of venom gland of Bothrops jararacussu. The full-length nucleotide sequence of 366 base pairs encodes a predicted gene product with 122 amino acid with theoretical isoelectric point and size of 5.28 and 13,685 kDa, respectively. This acidic PLA2 sequence was cloned into expression vector pET11a (+) and expressed as inclusion bodies in Escherichia coli BL21(DE3)pLysS. The N-terminal amino acid sequence of the 14 kDa recombinant protein was determined. The recombinant acidic PLA2 protein was submitted to refolding and to be purified by RP-HPLC chromatography. The structure and function of the recombinant protein was compared to that of the native protein by circular dichroism (CD), enzymatic activity, edema-inducing, and platelet aggregation inhibition activities.

Amino Acid Sequence↗

PubMed1993

Age- and tissue-dependent expression of CYP2C23 in the rat.

A cDNA was isolated from a library prepared with 15-day-old rat liver RNA and was shown to correspond to CYP2C23 with some minor alterations which modified the open reading frame. The expression of CYP2C23 was examined in rat liver, kidney, lung and testis and during ontogenesis. An appreciable amount of CYP2C23 RNA was observed in liver and kidney but the age-dependent expression was quite different between these two tissues. In liver, expression reached its maximal value in early neonates and remained quite stable, while the increase was progressive in kidney before declining after 3 weeks of age. In the liver, classical inducers of cytochrome P-450 decreased the CYP2C23 RNA content. Experimental data confirm the classification of CYP2C23 as a constitutive member of the 2C subfamily and clearly establish its age- and tissue-dependent expression in rat.

Aging↗

PubMed2000

Simultaneous expression of different immunogenic antigens in acute myeloid leukemia.

Identification of immunogenic leukemia-associated antigens as target structures is mandatory for specific immunotherapy of leukemia. Here, we define acute myeloid leukemia (AML) antigens eliciting a humoral immune response in the autologous host. We applied the method of serologic screening of cDNA expression libraries with autologous serum (SEREX). To date, this technique has been used to characterize antigen structures in solid tumors. The mRNA expression pattern of these newly in AML isolated antigens and previously described leukemia antigens (PRAME, MAGE-1, and Wt-1) was evaluated by reverse transcriptase polymerase chain reaction. For Wt-1, Western blotting also was performed. Screening of a cDNA expression library prepared from a patient with AML FAB M2 using autologous and allogeneic sera, followed by sequencing of positive clones, yielded three autoantigens (Prp1p/Zer1p, L19H1, and one without homology to previously described genes) and two antigens reactive with allogeneic sera (MAZ, PINCH). PRAME mRNA was expressed in 47% of 34 AML patients, but not in 13 CD34(+) cell samples or in peripheral blood mononuclear cells of 13 healthy volunteers. mRNA expression of MAZ was detected in 44% of AML patients, but only in 8% of healthy donors. Humoral responses to MAZ were detected in 35%. More than 80% of the screened AML patients showed simultaneous expression of two or more of these antigens.Differential expression in AML patients vs healthy volunteers suggests that the immunogenic antigens PRAME and MAZ are potential candidates for immunotherapy in AML.

Adaptor Proteins, Signal Transducing↗

PubMed1994

The very large amplifiable element AUD2 from Streptomyces lividans 66 has insertion sequence-like repeats at its ends.

In a spontaneous, chloramphenicol-sensitive (Cms), arginine-auxotrophic (Arg-) mutant of Streptomyces lividans 1326, two amplified DNA sequences were found. One of them was the well-characterized 5.7-kb ADS1 sequence, amplified to about 300 copies per chromosome. The second one was a 92-kb sequence called ADS2. ADS2 encoding the previously isolated mercury resistance genes of S. lividans was amplified to around 20 copies per chromosome. The complete ADS2 sequence was isolated from a genomic library of the mutant S. lividans 1326.32, constructed in the phage vector lambda EMBL4. In addition, the DNA sequences flanking the corresponding amplifiable element called AUD2 in the wild-type strain were isolated by using another genomic library prepared from S. lividans 1326 DNA. Analysis of the ends of AUD2 revealed the presence of an 846-bp sequence on both sides repeated in the same orientation. Each of the direct repeats ended with 18-bp inverted repeated sequences. This insertion sequence-like structure was confirmed by the DNA sequence determined from the amplified copy of the direct repeats which demonstrated a high degree of similarity of 65% identity in nucleic acid sequence to IS112 from Streptomyces albus. The recombination event leading to the amplification of AUD2 occurred within these direct repeats, as shown by DNA sequence analysis. The amplification of AUD2 was correlated with a deletion on one side of the flanking chromosomal region beginning very near or in the amplified DNA. Strains of S. lividans like TK20 and TK21 which are mercury sensitive have completely lost AUD2 together with flanking chromosomal DNA on one or both sides.

Amino Acid Sequence↗

PubMed1990

Preferential rearrangement of V kappa 4 gene segments in pre-B cell lines.

Examination of the in vitro V kappa gene rearrangements of murine adult bone marrow-derived pre-B cell lines reveals that 21 of 25 (84%) cell lines have rearranged a member of the V kappa 4 family. In contrast, analysis of two V kappa cDNA libraries prepared from LPS-stimulated adult spleen cells indicates that only 17% of the Ig kappa cDNAs contain sequences belonging to the V kappa 4 gene family. Half of the pre-B cell lines examined also share an 8-kbp BamHI reciprocal product (rp). However, these rp do not involve the same V kappa gene, indicating that conserved BamHI sites exist 3' of some V kappa genes. This rp is also readily detected in DNA from normal adult spleen cells, suggesting that the in vitro rearrangements examined in this study are representative of kappa rearrangements that occur in vivo. We suggest that, unlike the diverse V kappa repertoire expressed by mature B cells, the germline V kappa segments involved in initial rearrangements of the Ig kappa locus are highly restricted, and that an initial V kappa 4 rearrangement is probably followed by other, more random recombination events.

Animals↗

PubMed1990

Identification of a novel member (GDF-1) of the transforming growth factor-beta superfamily.

A cDNA clone encoding a new member (designated GDF-1) of the transforming growth factor-beta (TGF beta) superfamily was isolated from a library prepared from day 8.5 mouse embryos. The nucleotide sequence of GDF-1 predicts a protein of 357 amino acids with a mol wt of 38,600. The sequence contains a pair of arginine residues at positions 236-237, which is likely to represent a site for proteolytic processing. The C-terminus following the presumed dibasic cleavage site shows significant homology with the known members of the TGF beta superfamily, matching the other family members at all of the invariant positions, including the seven cysteine residues with their characteristic spacing. GDF-1 is most homologous to Xenopus Vg-1 (52%), but is not likely to be the murine homolog of Vg-1. In vitro translation experiments were consistent with GDF-1 being a secreted glycoprotein. Genomic Southern analysis indicated that GDF-1 may be highly conserved across species. These results suggest that GDF-1 is most likely an extracellular factor mediating cell differentiation events during embryonic development.

Amino Acid Sequence↗

PubMed2003

Two novel Bv8-like peptides from skin secretions of the toad Bombina maxima.

Two novel bioactive peptides were purified from skin secretions of the toad Bombina maxima. The partial N-terminal sequences of these two peptides were determined by automated Edman degradation. This allowed the cloning of full-length cDNAs encoding these two peptides from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences indicate that both peptides are composed of 77 amino acids. A FASTA search in the databanks revealed that they exhibit 86-91% sequence identity with Bv8, a peptide originally isolated from skin secretions of Bombina variegata. They were thus named as Bv8-like peptide 1 (Bv8-LP1) and Bv8-like peptide 2 (Bv8-LP2), respectively. Sequence differences between Bv8-LP1 and 2 were due to six amino acid substitutions at positions 6, 11, 23, 24, 62 and 63. Bv8-LP1 and 2 differed from Bv8 with eleven and seven amino acid substitutions, respectively. Like Bv8, Bv8-LP1 and 2 possessed contractile activity on isolated guinea pig ileum. Additionally, they stimulated contraction of rabbit aortic rings in a dose-dependent manner at nanomolar concentrations.

Amino Acid Sequence↗