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At least 685 records · Page 38Linked to original sources
PubMed1999

A bacterial single-chain Fv antibody fragment that inhibits binding of its parental anti-E-selectin monoclonal antibody to activated human endothelial cells.

Using the polymerase chain reaction, we cloned, modified, and linked antibody variable (V) region coding genes from a mouse hybridoma, and produced a bacterial single-chain Fv (scFv) antibody fragment specific for E-Selectin. A vector of pBR322 origin, bearing the tryptophan promoter and the ompA bacterial signal peptide, was used to direct scFv expression to periplasm. The vector included a six-histidine coding sequence 5' to the scFv for the purification of the expressed protein using immobilized metal affinity chromatography (IMAC). We found that the VH-Linker-VL 32-33 kDa scFv remained insoluble after cellular fractionation, and transmission electron microscopy showed the new protein to be present in the periplasm as inclusion bodies. The scFv was solubilized using urea, purified using IMAC, and renatured to its active form. In a competitive enzyme-linked immunosorbent assay with activated human vein endothelial cells in the solid phase, the scFv competed for binding with the original monoclonal antibody.

Amino Acid Sequence↗
PubMed1984

Androgen induction of ornithine decarboxylase mRNA in mouse kidney as studied by complementary DNA.

To investigate the mechanisms by which androgens regulate ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) in mouse kidney, a cDNA clone encoding OrnDCase mRNA was prepared. Purification of OrnDCase mRNA from kidneys of androgen-treated mice was accomplished by immunoadsorption of renal polysomes to a protein A-Sepharose column and enrichment for poly(A)-containing RNA by oligo(dT)-cellulose. Double-stranded cDNA synthesized from this mRNA was inserted into the Pst I site of plasmid pBR322 by using oligo(dG . dC)-tailing and was propagated in Escherichia coli. Plasmids containing cDNA sequences coding for OrnDCase were identified by differential colony hybridization, by radioimmunological detection of OrnDCase-like antigens in bacterial cultures, and by cell-free translation of hybrid-selected mRNA followed by immunoprecipitation with monospecific OrnDCase antiserum. A restriction endonuclease fragment of the selected plasmid DNA (pODC54) was labeled by nick-translation and used to study changes in OrnDCase mRNA concentration. After a single dose of testosterone, renal OrnDCase mRNA concentration increased as soon as 6 hr and peaked 24 hr after steroid injection, as measured by RNA blot hybridization. Continuous androgen treatment for 4 days resulted in a 10- to 20-fold increase in OrnDCase mRNA concentration in normal animals, but no induction of this mRNA was detected in mice that have an inherent defect of the androgen receptor (testicular feminization). These results indicate that androgens regulate OrnD-Case synthesis in mouse kidney, at least in part, by increasing OrnDCase mRNA accumulation.

Animals↗
PubMed1995

Expression of an Arabidopsis potassium channel gene in guard cells.

The Arabidopsis thaliana KAT1 cDNA encodes a voltage-gated inward-rectifying K+ channel. A KAT1 genomic DNA clone was isolated and sequenced, and a 5' promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KAT1 promoter fused to bacterial beta-glucuronidase showed robust beta-glucuronidase activity primarily in guard cells.

Arabidopsis↗
PubMed1987

Structure and evolution of the repetitive gene encoding streptococcal protein G.

The complete sequence of the structural gene encoding the immunoglobulin G binding protein from Streptococcus G148 has been determined, as well as its 5' and 3' flanking sequences. The sequence reveals an open reading frame encoding a putative preprotein with a relative molecular mass of 63294. N-Terminal sequencing of the mature protein, spontaneously released from streptococcal cells, demonstrates that the signal peptide consists of 33 amino acids. The DNA sequence reveals extensive internal homologies similar to other cell-wall-bound receptors from gram-positive bacteria. Comparisons with a related gene previously isolated from another strain of streptococci revealed large differences in size, due to variations in the number of internal repeats. The structure of the gene suggests an evolution through multiple duplications.

Amino Acid Sequence↗
PubMed2006

The gene overexpressed in polycythemia rubra vera, PRV-1, and the gene encoding a neutrophil alloantigen, NB1, are alleles of a single gene, CD177, in chromosome band 19q13.31.

BACKGROUND: PRV-1 mRNA is overexpressed by neutrophils from polycythemia vera patients and is homologous to NB1 a gene overexpressed in reactive neutrophilia. STUDY DESIGN AND METHODS: These investigations were designed to confirm searches of genome databases suggesting that PRV-1 and NB1 are alleles of the same gene, CD177, and confirm a pseudogene adjacent to CD177. Methods included polymerase chain reaction (PCR), cloning, sequencing, and fluorescent hybridization studies. RESULTS: The coding region of PRV-1 was PCR-amplified from human fetal RNA, cloned, and used to screen the RPCI-11 bacterial artificial chromosome (BAC) library. Five BACs were reactive with the PRV-1 probe. PCR analysis of the BACs with primers encompassing PRV-1 exons, containing four known single-nucleotide polymorphisms, followed by sequencing rendered amplicons identical to PRV-1 in all five BACs. Analysis of all five by restriction digestion yielded fragments possible only if both the gene and the pseudogene are present. End sequencing of the BACs localized them to the same chromosome region. G-banding and fluorescence in situ hybridization at the 400- and 850-band levels of resolution mapped one BAC to chromosome band 19q13.2 and sublocalized the BAC to band 19q13.31, respectively. CONCLUSION: PRV-1 and NB1 are alleles of the same gene now referred to as CD177. Changes in CD177 expression may be a marker of increased or decreased myelopoiesis and are therefore an effect of, rather than a cause of, myeloproliferative disorders.

Alleles↗
PubMed1996

Identification of the Streptococcus mutans frp gene as a potential regulator of fructosyltransferase expression.

Four putative open reading frames (ORFs) were previously identified in the regions flanking the Streptococcus mutans GS-5 fructosyltransferase (FTF) gene. One of these, ORF 3, appeared to code for a low-molecular-mass protein containing amino acid sequences sharing homology with several Gram-positive bacterial DNA-binding proteins and it was suggested that the ORF 3 gene product might be an FTF regulatory protein (FRP). In order to characterize this protein, we have purified the biotinylated tag-FRP fusion protein using the PinPoint protein purification system and this fusion protein was used in gel shift assays with DNA fragments containing the ftf promoter region. FRP bound specifically to the upstream region of the ftf promoter containing the inverted repeat structure that is present upstream of the -35 sequence. In contrast, FRP did not bind to DNA fragments lacking the inverted repeat structure. The results of these experiments suggest that FRP interacts with the inverted repeat region upstream of the ftf promoter and such interactions may regulate FTF expression.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of ssb and uup mutants with increased frequency of precise excision of transposon Tn10 derivatives: nucleotide sequence of uup in Escherichia coli.

A Lac+ papillation assay was used to identify mutants (tex) of Escherichia coli that exhibit an increased frequency of precise excision of a lacZ::Tn10dKan insertion. Three tex strains had suffered mutations in the gene (ssb) encoding the essential single-stranded DNA-binding protein SSB, which resulted in the following alterations in the 177-residue protein: G4D; L10F, P24S; and V102M. The phenotypes of these ssb mutants indicated that they were largely unaffected in other functions mediated by SSB, such as DNA replication, recombination, and repair. Strains with multicopy ssb+ exhibited a decreased frequency of Tn10dKan precise excision. Three other tex mutants had insertion mutations in the locus designated uup at 21.75 min on the linkage map. The nucleotide sequence of uup was determined, and the gene was inferred to encode a 625-amino-acid hydrophilic protein that belongs to the superfamily of ABC-domain proteins (with two pairs of the Walker A and B motifs), which are postulated to be involved in coupling ATP hydrolysis with other biological processes. The uup gene product shares extensive homology with the deduced sequences of two proteins of Haemophilus influenzae. The uup gene is also situated immediately upstream of (and is transcribed in the same direction as) the paraquat-inducible SoxRS-regulated pqi-5 gene, two reported promoters for which are situated within the uup coding sequence.

ATP-Binding Cassette Transporters↗
PubMed2006

[Construction of a recombinant adenovirus vector for interferon-gamma-inducible protein 10 and the adenovirus preparation].

OBJECTIVE: To construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination. METHODS: IP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein (EGFP). The shuttle plasmid was then transformed into E. coli BJ5183 with pAdEasy-1 vector by chemical transformation. The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells. RESULTS: The positive clones were identified with enzyme digestion and polymerase chain reaction (PCR) and were further verified by DNA sequencing. The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells. CONCLUSION: A recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein.

Adenoviridae↗
PubMed1989

Structural characterization of the BCR gene product.

Chronic myelogenous leukemia (CML) is associated with the reciprocal translocation of a region of chromosome 22 called BCR with the c-abl gene of chromosome 9.5' coding sequences from the BCR gene are spliced in-frame to the second exon of the ABL gene to produce a CML-specific 8.5 kilobase message which encodes the BCR-ABL hybrid protein P210. To definitively identify and characterize the normal BCR gene product, sequences from BCR cDNA clones were used to reconstitute the coding portion of the normal message in retroviral and bacterial transcription vectors. The normal BCR gene product was demonstrated to be a phosphoprotein of 160 kilodaltons by in vitro translation and immunoprecipitation from lysates of NIH3T3 lines expressing BCR retroviruses. Whereas BCR-homologous RNA levels in these cell lines were increased 50 fold, BCR protein levels increased only 2 to 10 fold depending on the presence or absence of BCR-specific 5' and 3' untranslated regions. We observe a kinase activity associated with this protein but we do not observe morphological transformation of NIH3T3 cells as a result of its overproduction.

Cloning, Molecular↗
PubMed1980

DNA sequences from the str operon of Escherichia coli.

The str operon at 72 min on the Escherichia coli chromosome contains genes for ribosomal proteins (r-proteins) S12 (str or rpsL) and S7 (rpsG) and elongation factors G (fus) and Tu (tufA). The sequence of the entire S12 gene, the S12-S7 intercistronic region, and the beginning of the S7 gene is reported. Also, the sequence of the end of the S7 gene, the S7-G intercistronic region, and the beginning of the elongation fractor G gene is reported. The S12-S7 intercistronic region is 96 base pairs long, in contrast to other intercistronic regions in r-protein operons which have been found to vary from 3 to 66 base pairs. The S7-G intercistronic region is only 27 bases long, supporting the previous conclusion that r-protein and elongation factor genes are co-transcribed. A comparison of translation initiation sites of the S12 and S7 genes, and other examples of co-transcribed r-protein genes, reveals no obvious features that could account for equimolar synthesis of all r-proteins. The codon usage in the S12 and S7 genes follows the pattern observed in other r-protein genes; that is, there is a highly preferential usage of codons recognized by the most abundant of isoaccepting tRNA species. This pattern could reflect the cell's need for efficient translation or minimal errors, or both, in r-protein synthesis.

Amino Acid Sequence↗
PubMed2002

Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli.

Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.

Adrenal Glands↗