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At least 235 records · Page 13Linked to original sources
PubMed1995

Characterization of an avian bone sialoprotein (BSP) cDNA: comparisons to mammalian BSP and identification of conserved structural domains.

Bone sialoprotein (BSP) is one of the major noncollagenous proteins found in mineralized vertebrate tissue. It is an acidic glycoprotein containing a high sialic acid content and is phosphorylated on several of its Ser and Thr residues. While it has been extensively characterized from various mammalian species, little is known about its sequence or expression in lower vertebrates. The cloning and characterization of several cDNAs encoding the chicken bone sialoprotein are reported here. A partial cDNA clone encoding the carboxyl terminus of the protein was initially isolated from a lambda GT11 expression library using a polyclonal antibody gains BSP purified from chicken bone matrix. Subsequently, several additional clones were obtained by further screening and by reverse transcription polymerase chain reaction (RT-PCR). Three overlapping clones encompassing about 1 kb, which included the complete coding sequence for BSP, were analyzed. The deduced amino acid sequence revealed that chicken BSP contains 276 amino acid residues. Although the overall identity between chicken and mammalian BSP is only approximately 39%, the diversity in amino acid sequence occurs mostly between the major functional domains of this molecule. These domains include: (1) three acidic poly-Glu regions; (2) two tyrosine-rich domains, which may be sites for protein sulfation; (3) several casein kinase II phosphorylation sites; (4) an Asn glycosylation site; and (5) an RGD cell-binding motif. Of interest in the chicken BSP is the identification of two additional RGD motifs within the avian sequence, unlike the mammalian forms of BSP which has only one.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2004

Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma.

BACKGROUND: Disease relapse is the leading cause of mortality for children diagnosed with disseminated neuroblastoma. The adoptive transfer of tumor-specific T cells is an attractive approach to target minimal residual disease following conventional therapies. We describe here the genetic engineering of human cytotoxic T lymphocytes (CTL) to express a chimeric immunoreceptor for re-directed HLA-independent recognition of neuroblastoma. METHODS: The CE7R chimeric immunoreceptor was constructed by PCR splice overlap extension and is composed of a single-chain antibody extracellular domain (scFv) derived from the L1-CAM-specific murine CE7 hybridoma fused to human IgG1 hinge-Fc, the transmembrane portion of human CD4, and the cytoplasmic tail of huCD3-zeta chain (scFvFc:zeta). Primary human T cells were genetically modified by naked DNA electrotransfer of plasmid expression vector CE7R-pMG then analyzed by Western blotting, flow cytometry for CE7R expression and cell surface trafficking, 4-h chromium release assay for re-directed neuroblastoma lysis, and ELISA for tumor-specific activation of cytokine production. RESULTS: CE7R is expressed as an intact chimeric protein that trafficks to the cell surface as a type I transmembrane protein. Primary human CE7R-expressing CD8(+) CTL clones specifically recognize human neuroblastoma tumor cells and are activated for tumor cell lysis and T(c)1 cytokine production. CONCLUSIONS: These data demonstrate the utility of CE7R for re-directing the effector function of CTL to neuroblastoma and have provided the rationale to initiate a FDA-authorized (BB-IND#9149) pilot clinical trial to establish the feasibility and safety of adoptive transfer of autologous CE7R(+)CD8(+) CTL clones to children with recurrent/refractory neuroblastoma.

CD3 Complex↗
PubMed2003

Mutation analysis of the feedback inhibition site of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli.

In Escherichia coli, the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) AroG catalyzes the first committed step in the biosynthesis of aromatic compounds. To investigate the feedback inhibition site of AroG, mutated enzymes prepared with sequence-overlap extension PCR were expressed and purified. The enzymatic activity assay showed that the amino acid replacements at Phe144, Leu175, Leu179, Phe209, Trp215Ala and Val221 completely or partially relieved feedback inhibition of AroG addressed by the phenylalanine. Ile10Ala and Delta(1-15) desensitized feedback inhibition and caused a 70 approximately 90% loss of the specific catalytic activities. These results strongly suggest an involvement of the interior region and the N-terminus of the polypeptide chain of AroG in the formation of the feedback inhibition site of DAHPS.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1996

Recombinant alpha 1(VIII) collagen chains form homotrimers in vitro.

Type VIII collagen, a member of the short chain collagen family, is expressed in large blood vessels and in the subendothelium and mesangium of the glomerulus. While two genetically distinct type VIII collagen chains, alpha 1 and alpha 2, have been identified, it is not known whether type VIII collagen exists as homotrimers or heterotrimers. To determine whether alpha 1(VIII) collagen chains can associate to form homotrimers, a full length human alpha 1(VIII) collagen cDNA was generated by overlap extension PCR and used as a substrate for coupled in vitro transcription/translation. A translation product of 80kDa, the predicted size of alpha 1(VIII) collagen, was identified by autoradiography of SDS-gels containing radiolabelled translation products. Sensitivity of the in vitro translated protein to digestion with bacterial collagenase and trypsin and the size of the proteins generated by these digestions provide evidence that alpha 1(VIII) collagen can participate in the formation of homotrimers.

Biopolymers↗
PubMed1998

An autoantibody is modified for use as a delivery system to target the cell nucleus: therapeutic implications.

A murine monoclonal anti-dsDNA antibody was found to penetrate living cells and localize in the nucleus without pathologic effects. A single mutation in VH markedly enhanced cellular penetration. The mutant antibody was produced as recombinant Fab and single chain antibody fragments to investigate its use as a delivery system to target the cell nucleus. Complexes were made containing Fab fragments and alkaline phosphatase conjugated goat antibodies to mouse |gk chains. Fab fragments transported 305 kDa goat antibody-enzyme complexes into the nucleus in COS-7 and CHO cells. A single chain antibody cDNA was constructed by splice overlap extension PCR and expressed in COS-7 cells. Binding of the single chain antibody to dsDNA was shown by ELISA, and cellular penetration and nuclear localization were demonstrated in COS-7 and CHO cells. The single chain antibody cDNA was ligated into the expression vector, pEGFP, to produce a fusion protein with green fluorescent protein. The fusion protein penetrated COS-7 cells and localized in the cell nucleus. The single chain antibody produced during sustained expression in CHO cells re-entered antibody-producing cells and localized in the nucleus without affecting cell viability. Our results demonstrate the potential use of a modified autoantibody as a delivery system to target the cell nucleus.

Animals↗
PubMed1999

The complete genomic sequence of an HTLV-II isolate from a Guahibo Indian from Venezuela.

A polyclonal CD3(+), CD8(+) T-cell line, G2, was derived from the peripheral blood of a seropositive, PCR-positive, HTLV-IIB infected Guahibo Indian from Venezuela. The cell line is productively infected with HTLV-IIB. The entire HTLV-II G2 proviral DNA was sequenced via PCR using overlapping HTLV-II primer pairs. Phylogenetic analyses indicate that HTLV-II G2 is the most divergent HTLV-IIB strain identified to date. Characterization of its deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

Amino Acid Sequence↗
PubMed2007

Identification of three Zwittermicin A biosynthesis-related genes from Bacillus thuringiensis subsp. kurstaki strain YBT-1520.

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.

Bacillus thuringiensis↗
PubMed2006

Co-occurrence of both L-asparaginase subtypes in Arabidopsis: At3g16150 encodes a K+-dependent L-asparaginase.

L-asparaginases (EC 3.5.1.1) are hypothesized to play an important role in nitrogen supply to sink tissues, especially in legume-developing seeds. Two plant L-asparaginase subtypes were previously identified according to their K(+)-dependence for catalytic activity. An L-asparaginase homologous to Lupinus K(+)-independent enzymes with activity towards beta-aspartyl dipeptides, At5g08100, has been previously characterized as a member of the N-terminal nucleophile amidohydrolase superfamily in Arabidopsis. In this study, a K(+)-dependent L-asparaginase from Arabidopsis, At3g16150, is characterized. The recombinants At3g16150 and At5g08100 share a similar subunit structure and conserved autoproteolytic pentapeptide cleavage site, commencing with the catalytic Thr nucleophile, as determined by ESI-MS. The catalytic activity of At3g16150 was enhanced approximately tenfold in the presence of K(+). At3g16150 was strictly specific for L-Asn, and had no activity towards beta-aspartyl dipeptides. At3g16150 also had an approximately 80-fold higher catalytic efficiency with L-Asn relative to At5g08100. Among the beta-aspartyl dipeptides tested, At5g08100 had a preference for beta-aspartyl-His, with catalytic efficiency comparable to that with L-Asn. The phylogenetic analysis revealed that At3g16150 and At5g08100 belong to two distinct subfamilies. The transcript levels of At3g16150 and At5g08100 were highest in sink tissues, especially in flowers and siliques, early in development, as determined by quantitative RT-PCR. The overlapping spatial patterns of expression argue for a partially redundant function of the enzymes. However, the high catalytic efficiency suggests that the K(+)-dependent enzyme may metabolize L-Asn more efficiently under conditions of high metabolic demand for N.

Amino Acid Sequence↗
PubMed2005

Recombinant extracellular matrix-like proteins with repetitive elastin or collagen-like functional motifs.

Using overlap elongation PCR, we created repetitive DNA libraries encoding the elastin VPGVG and collagen-like GERGDRGDP sequences. From these libraries we isolated two repetitive DNA sequences, Col-5 encoding [(GERGDRGDP)(5)GER], and Ela-16 encoding [(VPGVG)(16)VPG]. Both proteins were expressed as thioredoxin fusion proteins. The resulting recombinant extracellular matrix-like proteins had the expected properties (cell adhesive ability and thermally responsive structural change) of the functional motif sequence unit used.

Amino Acid Motifs↗
PubMed1994

Activity of the E75E76 mutant of the alpha subunit of casein kinase II from Xenopus laevis.

The cDNA gene coding for the alpha subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation substituted glutamic acids for Lys75 and Lys76, changing the charge distribution of a very basic sequence found in the alpha subunit. Expression of the mutated cDNA in a pT7-7 vector in E. coli yielded an active mutant recombinant protein that was extensively purified. This mutant was not significantly affected in its app. Km for casein or a model peptide substrate, nor in its interaction with the activating beta subunit. Inhibition by quercetin and by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII alpha E75E76 mutant was at least one order of magnitude less sensitive to inhibition by polyanionic inhibitors such as heparin, poly U, copolyglutamic acid:tyrosine (4:1) and 2,3 diphosphoglycerate.

Amino Acid Sequence↗
PubMed2002

Development of an assay for beta-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein.

An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, beta-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and beta-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. beta-Lactamase catalyzes the hydrolysis of the beta-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the beta-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of beta-lactamase in a format that should be amenable to high-throughput screening.

Aequorin↗
PubMedDate not supplied

Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma.

The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma cell line FMC63 which produces monoclonal antibody specific for CD19. The genes encoding the FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material showed that the scFv binds target cells with a Ka of 2.3 x 10(-9), compared with 4.2 x 10(-9) for the parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy and immunogenicity, with a view to application in the diagnosis and treatment of human B cell cancers.

Animals↗
PubMed1998

Total chemical synthesis of the 3' untranslated region of the hepatitis C virus with long oligodeoxynucleotides.

Hepatitis C Virus (HCV) is the major causative agent of chronic hepatitis. Since it has been difficult to obtain full-length cDNA clones of HCV including the 3' untranslated region (UTR) that give rise to replication competent virus, we generated the 3'UTR by a modified protocol of total chemical synthesis (TCS) with overlap-extension-PCR using four long oligodeoxynucleotides. A synthetic cDNA fragment of about 340 nucleotides (nt) in length was generated, subcloned and sequenced. This approach represents a rapid and easy alternative to RT-PCR from infectious serum and may be a highly valuable method to generate partial cDNA clones of HCV and other viruses including defined variants.

3' Untranslated Regions↗
PubMed1993

Molecular cloning and expression of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase and sequence similarity with protein phosphatase 2C.

After many unsuccessful attempts to detect cDNA encoding the catalytic subunit of bovine pyruvate dehydrogenase phosphatase (PDPc) in bovine cDNA libraries, an approach based on the polymerase chain reaction (PCR) was undertaken. Overlapping DNA fragments were generated by PCR from bovine genomic DNA and from cDNA synthesized from total RNA with synthetic oligonucleotide primers on the basis of experimentally determined amino acid sequences. The DNA fragments were subcloned and sequenced. The complete cDNA is 1900 base pairs in length and contains an open reading frame of 1614 nucleotides encoding a putative presequence of 71 amino acid residues and a mature protein of 467 residues with a calculated M(r) of 52,625. Hybridization analysis showed a single mRNA transcript of about 2.0 kilobases. Comparison of the deduced amino acid sequences of the mitochondrial PDPc and the rat cytosolic protein phosphatase 2C indicates that these protein serine/threonine phosphatases evolved from a common ancestor. The mature form of PDPc was coexpressed in Escherichia coli with the chaperonin proteins groEL and groES. The recombinant protein (rPDPc) was purified to near homogeneity. Its activity toward the bovine 32P-labeled pyruvate dehydrogenase complex was Mg(2+)-dependent and Ca(2+)-stimulated and comparable to that of native bovine PDP. An active, truncated form of rPDPc, with M(r) approximately 45,000, was produced in variable amounts during growth of cells and/or during the purification procedure.

Amino Acid Sequence↗
PubMed2006

Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli.

A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45 degrees C, respectively, and the enzyme maintained high activity at pH 5.0 and 60 degrees C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (k(cat)/K(M)) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached approximately 71% at 20 degrees C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for alpha-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.

Amino Acid Sequence↗
PubMed1997

Enhanced expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage.

The gene encoding green fluorescent protein (GFP) from Aequorea victoria was resynthesized to adapt its codon usage for expression in plants by increasing the frequency of codons with a C or a G in the third position from 32 to 60%. The strategy for constructing the synthetic gfp gene was based on the overlap extension PCR method using 12 long oligonucleotides as the starting material and as primers. The new gene contains 101 silent nucleotide changes compared to its wild-type counterpart used in this study. Several transgenic tobacco lines containing the wild-type gfp gene contained minute amounts of a smaller protein cross-reacting with GFP antiserum, whereas only one protein of the expected size was found in transgenics with the synthetic gfp gene. The smaller protein was probably encoded by a truncated gfp mRNA created by splicing of a 84 bp cryptic intron as detected by a reverse transcription-PCR technique. A comparison of GFP production in transgenics with the wild-type and the synthetic gfp gene under the control of the enhanced CaMV 35S promoter showed that the large-scale alterations in the gfp gene increased the frequency of high expressors in the transgenic population but hardly changed the maximum GFP concentrations. The latter phenomenon may be attributed to a reduced regeneration capacity of transformed cells with higher GFP concentrations.

Amino Acid Sequence↗
PubMed2002

Identification of a region of homozygous deletion on 8p22-23.1 in medulloblastoma.

To identify critical tumor suppressor loci that are associated with the development of medulloblastoma, we performed a comprehensive genome-wide allelotype analysis in a series of 12 medulloblastomas. Non-random allelic imbalances were identified on chromosomes 7q (58.3%), 8p (66.7%), 16q (58.3%), 17p (58.3%) and 17q (66.7%). Comparative genomic hybridization analysis confirmed that allelic imbalances on 8p, 16q and 17p were due to loss of genetic materials. Finer deletion mapping in an expanded series of 23 medulloblastomas localized the common deletion region on 8p to an interval of 18.14 cM on 8p22-23.2. We then searched within the region of loss on 8p for loci that might contain homozygous deletion using comparative duplex PCR. An overlapping homozygous deletion region was identified in a 1.8 cM interval on 8p22-23.1, between markers D8S520 and D8S1130, in two medulloblastomas. This region of homozygous deletion also encompasses the 1.4 cM minimal deletion region detected on 8p in ductal carcinoma in situ of breast. In conclusion, we reported for the first time a detailed deletion mapping on 8p in medulloblastoma and have identified a region of homozygous deletion on 8p22-23.1 that is likely to contain a critical tumor suppressor gene involved in the development of medulloblastoma.

Adolescent↗
PubMed2003

Identification of an anion-specific channel in the cell wall of the Gram-positive bacterium Corynebacterium glutamicum.

A cation-selective channel (porin), designated PorA, facilitates the passage of hydrophilic solutes across the cell wall of the mycolic acid-containing actinomycete Corynebacterium glutamicum. Biochemical and electrophysiological investigations of the cell wall of the mutant strain revealed the presence of an alternative channel-forming protein. This porin was purified to homogeneity and studied in lipid bilayer membranes. It forms small anion-selective channels with a diameter of about 1.4 nm and an average single-channel conductance of about 700 pS in 1 M KCl. The PorBCglut channel could be blocked by citrate in a dose-dependent manner. This result was in agreement with growth experiments in citrate as sole carbon source where growth in citrate was impaired as compared with growth in other carbon sources. The PorBCglut protein was partially sequenced and based on the resulting amino acid sequence of the corresponding gene, which was designated as porB, was identified as an unannotated 381 bp long open reading frame (ORF) in the published genome sequence of C. glutamicum ATCC13032. PorBCglut contains 126 amino acids with an N-terminal extension of 27 amino acids. One hundred and thirty-eight base pairs downstream of porB, we found an ORF that codes for a protein with about 30% identity to PorBCglut, which was named PorCCglut. The arrangement of porB and porC on the chromosome suggested that both genes belong to the same cluster. RT-PCR from overlapping regions between genes from wild-type C. glutamicum ATCC 13032 and its ATCC 13032DeltaporA mutant demonstrated that this is the case and that porB and porC are cotranscribed. The gene products PorBCglut and PorCCglut represent obviously other permeability pathways for the transport of hydrophilic compounds through the cell wall of C. glutamicum.

Amino Acid Sequence↗