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At least 847 records · Page 47Linked to original sources
PubMed1990

The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II.

The entire coding sequence of wild-type mouse p53 was expressed in Escherichia coli under control of the PL promoter of bacteriophage lambda. The bacterial p53 protein had identical mobility to p53 from SV3T3 cells on SDS polyacrylamide gels and was recognized in bacterial lysates by three p53-specific monoclonal antibodies, including PAb246 which is specific for wild-type mouse p53. Immunoprecipitates of the bacterial p53 were phosphorylated by a highly purified preparation of rat casein kinase II; the stoichiometry of incorporation was approximately 1 mol of phosphate per mol of p53. The phosphorylated residue was identified by phosphopeptide mapping as serine 389, which is a major site of p53 phosphorylation in vivo. p53 (serine 389) kinase activity was detected on lysates of SV3T3 cells; this activity co-purified with casein kinase II on phosphocellulose and Mono Q columns and was inhibited by heparin. Immunoprecipitates of the p53-T antigen complex from SV3T3 cells also had associated serine 389 kinase activity. Phosphorylation of serine 389 by this kinase was potently inhibited by heparin and quenched by excess unlabelled GTP. The data indicate that p53 is a physiological substrate of casein kinase II, which is stimulated in response to mitogens, phosphorylates nuclear oncoproteins, and may play a role in the transduction of extracellular signals to the nucleus.

Amino Acid Sequence↗
PubMed1985

The common alpha subunit of bovine glycoprotein hormones: limited formation of native structure by the totally nonglycosylated polypeptide chain.

The folding of the bovine glycoprotein hormone alpha subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process. The bacterially derived alpha polypeptide (bac-alpha), extracted from E. coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay. Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18-fold. Approximately 2% of the refolded bac-alpha preparation combines with the beta subunit of human chorionic gonadotropin (hCG beta) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac-alpha (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1%), it appears that correct folding of alpha subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin alpha subunit (LH alpha) and chemically deglycosylated LH alpha (which retains two asparagine-linked N-acetyl glucosamine residues per alpha oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced alpha subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LH alpha fully regained its native structure. The partially deglycosylated LH alpha also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HCG beta to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional alpha subunit.

Animals↗
PubMed2006

Specific selection pressure at the third codon positions: contribution to 10- to 11-base periodicity in prokaryotic genomes.

Prokaryotic sequences are responsible for more than just protein coding. There are two 10- to 11-base periodical patterns superimposed on the protein coding message within the same sequence. Positional auto- and cross-correlation analysis of the sequences shows that these two patterns are a short-range counter-phase oscillation of AA and TT dinucleotides and a medium-range in-phase oscillation of the same dinucleotides, spanning distances of up to approximately 30 and approximately 100 bases, respectively. The short-range oscillation is encoded by the amino acid sequences themselves, apparently, due to the presence of amphipathic alpha-helices in the proteins. The medium-range oscillation, related to DNA folding in the cell, is created largely by a special choice of the bases in the third positions of the codons. Interestingly, the amino acid sequences do contribute to that signal as well. That is, the very amino acid sequences are, to some extent, degenerate to serve the same oscillating pattern that is associated with the degenerate third codon positions.

Amino Acid Sequence↗
PubMed1995

A murine antibody to Shigella dysenteriae type 1 employs V-genes that contain a rearranged codon for the lambda light chain.

The cDNA coding for a hybridoma anti Shigella dysenteriae type 1 antibody (3707 E9) has been cloned, and sequenced. Binding patterns with fragments of the bacterial polysaccharide antigen had already been studied in detail. The VH sequence utilizes the VH441 gene, first identified amongst beta-(1,6)galactan-binding antibodies, while the VL is closely related to the V lambda 1 gene. We found that the VL3707 E9 gene employed a VL-J combinatorial joining leading to a rare Trp-->Leu substitution at position L96.

Amino Acid Sequence↗
PubMed1982

DNA sequences flanking an E. coli insertion element IS2 in a cloned yeast TRP5 gene.

The insertion of an Escherichia coli IS2 element upstream from a cloned yeast TRP5 gene results in an increased level of active tryptophan synthase in trpAB E. coli host cells. This insertion occurs about 60 bp upstream from the first AUG of the TRP5 gene and is associated with a duplication of the sequence TTACA at the target site. The nucleotide sequence corresponding to the first 173 amino acids of the yeast TRP5 gene has also been determined. The N-terminal region of the yeast tryptophan synthase includes areas of strong homology with the alpha-subunit of the corresponding E. coli enzyme. Sequences from the 5' untranslated region upstream from the TRP5 gene are compared to homologous areas of other yeast genes.

Base Sequence↗
PubMed2001

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries.

We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.

Amino Acid Sequence↗
PubMed2000

Gene organization of a Plasmodium falciparum serine hydroxymethyltransferase and its functional expression in Escherichia coli.

The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Three reactions are involved in methylenetetrahydrofolate recycling: Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). Malarial bifunctional DHFR-TS is a well-studied, important target of established drugs such as pyrimethamine and cycloguanil. In sharp contrast, malarial SHMT remains largely uncharacterized. In the present study, a Plasmodium falciparum SHMT coding region was characterized. It had 1603 bp including two introns near the 5'-end of the gene: one 118 bp intron immediately after the start methionine and a 159 bp intron after an additional 34 amino acids. The three exons together coded for a 442 amino acid protein with 38-47% identity to SHMT sequences from other species. Expression of malarial SHMT coding sequence (minus the introns) into glyA mutants of Escherichia coli relieved glycine auxotrophy and permitted direct assay of SHMT catalytic activity in bacterial cell lysates. This is the first SHMT cloned and expressed from a protozoan parasite. The molecular tools developed in this study will be useful for developing potential antimalarials directed at SHMT.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.

Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins.

Amino Acid Sequence↗
PubMed1991

High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme.

A rat cDNA containing the complete coding sequence for rat tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) was isolated from a rat PC12 cDNA library and subcloned in a bacterial expression plasmid, and large amounts of functional enzyme were produced in Escherichia coli. The recombinant enzyme was purified approximately 20-fold to a final specific activity of 1.8 mumol/min per mg of protein, with a yield of 30%. As much as 1 mg of pure protein could be obtained from 1 g of wet bacterial cells. The purified hydroxylase was shown to be homogeneous by denaturing polyacrylamide electrophoresis and isoelectric focusing. Amino acid analysis of the N terminus (25 residues) revealed 100% identity with rat PC12 tyrosine hydroxylase, as deduced from its cDNA sequence. Several of the kinetic properties of the recombinant enzyme resembled those of the native PC12 hydroxylase. However, in contrast to the native enzyme, the purified recombinant hydroxylase was shown to be in an activated form. Phosphorylation with cAMP-dependent protein kinase resulted in stoichiometric incorporation of phosphate, but the kinetic profile of the recombinant enzyme was unaffected. Several clues to these differences are considered that may provide insight into the structural features important to the regulation of tyrosine hydroxylase.

Adrenal Gland Neoplasms↗
PubMed1991

Isolation and cDNA cloning of a rat O6-alkylguanine-DNA-alkyltransferase gene, molecular analysis of expression in rat liver.

A rat O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA has been isolated from a rat liver cDNA library by hybridization with the human homologue. The candidate 806 bp cDNA was sequenced and shown to contain a 630 bp open reading frame that could encode a protein of 22.2 kd. Fluorography of labelled ATase indicates a 24 kd protein which is consistent with several previous reports. The derived amino acid sequence demonstrated 81% similarity with the human ATase and 94% identity over a 67 residue region encompassing the putative alkyl acceptor site. Peptide sequences derived from cleaved homogeneous rat ATase have been located in the predicted protein providing additional confirmation of the identity of the cDNA. A 1.05 kb mRNA has been detected in rat liver by Northern analysis; treatment of adult rats with 2-acetylaminofluorene causes an approximately 10-fold induction of this message in liver. Following site directed mutagenesis of the 806 bp cDNA, the 630 bp protein coding sequence has been ligated into an Escherichia coli expression vector to achieve ATase levels of greater than 3% of total protein in bacterial extracts.

2-Acetylaminofluorene↗