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J G Files

Publications and source records attributed to J G Files.

13 recordsLinked to original sources
PubMed1998

A novel sensitive and selective bioassay for human type I interferons.

We describe a novel MxA gene-induction assay for type I interferons (IFN-alpha and IFN-beta) based on the specific induction of the MxA gene in cultured human cells. Accumulated intracellular MxA protein is determined by immunologic measurement by a rapid method using commercially available materials. IFN activity can be measured accurately over a concentration range of 0.1-30 IU/ml. In contrast, type II IFN and other cytokines are not significantly detected. The MxA-induction assay has advantages in terms of specificity, reliability, and sensitivity over other methods for assaying type I IFN. It has also been adapted and validated for measuring the titers of anti-IFN-beta neutralizing antibodies in human sera.

Antiviral Agents↗
PubMed1998

A novel bioassay for the determination of neutralizing antibodies to IFN-beta1b.

We have adapted the new MxA gene-induction bioassay to measure neutralizing antibodies to interferon-beta1b (IFN-beta1b, the active ingredient in Betaseron) in sera from patients treated with Betaseron. This antibody assay has been validated to quantify neutralizing titers of 1:20 and above, with a precision of +/- 0.20 in log10. We have used this MxA gene-induction antibody assay to reinvestigate serum samples from multiple sclerosis (MS) patients treated with Betaseron. The titers measured were closely comparable to those obtained in antiviral assays. Data obtained by both methods show that neutralizing antibodies may appear and subsequently disappear over time in the sera of some patients treated with Betaseron. Sera from some patients contain binding antibodies to IFN-beta1b. It was shown that binding antibody titers do not correlate quantitatively or qualitatively with neutralizing antibody titers, and indeed, a number of patients develop high levels of binding antibodies but never form measurable levels of neutralizing antibodies.

Antigen-Antibody Reactions↗
PubMed1988

Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli.

An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12. The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge. De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation. A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated. The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides. The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein. The products synthesized in E. coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite. The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.

Amino Acid Sequence↗
PubMed1983

Actin gene family of Caenorhabditis elegans.

Four actin genes have been isolated from Caenorhabditis elegans that account for all of the major actin hybridization to total genomic DNA. Actin genes I, II and III are clustered within a 12 X 10(3) base region; gene IV is unlinked to the others. All four genes have been sequenced from at least nucleotide -109 to +250. Genes I and III are identical for the first 307 coding nucleotides. Genes I and II differ in 14 positions within the first 250 coding nucleotides; one difference substitutes an aspartic acid for a glutamic acid at codon 5. Genes I and IV differ in 18 positions within the first 259 coding nucleotides without causing any amino acid differences. Genes I, II and III have introns after the first nucleotide of codon 64 and gene IV has an intron between codons 19 and 20. The four nucleotide sequences thus far define two different amino acid sequences. Both of the amino acid sequences resemble vertebrate cytoplasmic actin more than vertebrate muscle actin. A DNA polymorphism between the Bristol and Bergerac strains has been used as a phenotypic marker in genetic crosses to map the cluster of actin genes within a 2% recombination interval on linkage group V between unc-23 and sma-1 in order to begin a molecular genetic analysis of the actin loci.

Actins↗
PubMed1976

Limited proteolytic digestion of lac repressor by trypsin. Chemical nature of the resulting trypsin-resistant core.

Tryptic digestion of Escherichia coli lac repressor under nondenaturing conditions readily removes 59 amino acids from the NH2-terminal end of the polypeptide chain. Longer digestion removes an additional 20 or more amino acids from the COOH terminus, leaving a highly trypsin-resistanct core molecule. The lac repressor tetrameric structure and inducer-binding activity are retained by the tryptic core. Operator-binding activity, however, is lost as the NH2-terminal end is degraded. Many or all of the possible trypsin cleavage sites in the NH2-terminal region are available to attack by the enzyme, indicating that this part of the polypeptide chain is exposed to the environment. Lac repressor, missing the NH2-terminal end, renatures efficiently from random-coil solvent to tetramers with full inducer-binding activity, indicating that the NH2-terminal region is not necessary for the appropriate three-dimensional folding of the polypeptide chains. Core which lacks both the NH2 and COOH termini renatures to tetramers with low efficiency.

Amino Acid Sequence↗
PubMed1974

Translational reinitiation: reinitiation of lac repressor fragments at three internal sites early in the lac i gene of Escherichia coli.

Three early amber mutations in the lac i gene have been shown to arise from the codons corresponding to residues 7, 12, and 17 of the lac repressor polypeptide chain. All three mutations allow translational reinitiation at the same two sites, resulting in the synthesis of two lac repressor fragments. The amino-terminal sequences of these fragments show that the first site is the triplet coding for valine residue 23, while the second is the first internal in-phase AUG codon corresponding to residue 42. Translational reinitiation appears to be a common event in E. coli, since there are at least three such sites in the first 70 in-phase codons of the i-gene messenger RNA, and all amber mutants found in this region show translational reinitiation. Only one of these sites involves an AUG codon; the other two involve an in vivo ambiguity of the genetic code, in that the same codon can be translated into two different amino acids depending on whether it is recognized during initiation or elongation of protein biosynthesis. The two non-AUG codons are the codons corresponding to leucine residue 62 and valine residue 23 of the lac repressor.

Amino Acid Sequence↗
PubMed1973

Reinitiation of a lac repressor fragment at a codon other than AUG.

52 Spontaneous nonsense mutants in the lac i gene of Escherichia coli were isolated and characterized. All mutants located early in the gene show negative complementation in vivo with a wild-type i gene in a recA diploid strain. In vitro studies show that those mutants that display negative complementing activity in vivo also make lac repressor fragments retaining inducer binding and immunological crossreactivity with wild-type repressor. Amino-acid sequence analysis of these fragments shows that they arise by reinitiation at internal sities of the i message after chain termination at a prior amber or ochre codon. There are at least two different internal reinitiation sites in the first 200 nucleotides of the translated part of the i message. The first site corresponds to the first internal in phase AUG codon, which specifies the methionine residue at position 42 of the repressor protein. This site can be activated by an amber codon, 45 nucleotides before the AUG codon. The second site is only 60 nucleotides past the first site and can be activated by an amber mutation derived from residue 60 of the protein. The second initiation codon specifies the amino-acid leucine in the wild-type repressor, but the reinitiated fragment shows an amino-terminal methionine residue at this position. Therefore, the second initiation site seems to involve an in vivo ambiguity of the genetic code in that the same codon can be translated into two different amino acids depending on the recognition of this codon during initiation (when methionine is inserted) or elongation of protein synthesis (when leucine is inserted). The possibility that a codon other than AUG can act as an initiation codon in vivo is discussed.

Adenine Nucleotides↗