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C G Miyada

Publications and source records attributed to C G Miyada.

At least 19 recordsLinked to original sources
PubMed1999

Mycobacterium species identification and rifampin resistance testing with high-density DNA probe arrays.

Species identification within the genus Mycobacterium and subsequent antibiotic susceptibility testing still rely on time-consuming, culture-based methods. Despite the recent development of DNA probes, which greatly reduce assay time, there is a need for a single platform assay capable of answering the multitude of diagnostic questions associated with this genus. We describe the use of a DNA probe array based on two sequence databases: one for the species identification of mycobacteria (82 unique 16S rRNA sequences corresponding to 54 phenotypical species) and the other for detecting Mycobacterium tuberculosis rifampin resistance (rpoB alleles). Species identification or rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Seventy mycobacterial isolates from 27 different species and 15 rifampin-resistant M. tuberculosis strains were tested. A total of 26 of 27 species were correctly identified as well as all of the rpoB mutants. This parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.

Antibiotics, Antitubercular↗
PubMed1996

Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays.

We have combined photochemistry and photolithography with solid-phase DNA synthesis chemistry to form a new technology that makes high density oligonucleotide probe array synthesis possible. Hybridization to these two-dimensional arrays containing hundreds or thousands of oligonucleotide probes provides a powerful DNA sequence analysis tool. Two types of light-generated DNA probe arrays have been used to test for a variety of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. One array, made up of 428 probes, was designed to scan through the length of CFTR exon 11 and identify differences from the wild type reference sequence. The second type of array contained 1480 probes chosen to detect known deletions, insertions, or base substitution mutations. The validity of the probe arrays was established by hybridizing them with fluorescently labeled control oligonucleotide targets. Characterized mutant CFTR genomic DNA samples were then used to further test probe array hybridization specificity. Finally, ten unknown patient samples were genotyped using the CFTR probe array assay. The genotype assignments were identical to those obtained by PCR product restriction fragment analysis. Our results show that light-generated DNA probe arrays are highly effective in analyzing complex mutation and polymorphism patterns in a relatively large gene such as CFTR.

Base Sequence↗
PubMed1995

Isolation, characterization and expression of the gene that encodes D-arabinitol dehydrogenase in Candida tropicalis.

The gene (ARD) that encodes NAD-dependent D-arabinitol dehydrogenase (ArDH) in the pathogenic fungus Candida tropicalis (Ct) was cloned by transforming Escherichia coli (Ec) BW31M (araCc) with a plasmid library of Ct genomic DNA and selecting for D-arabinitol-utilizing (D-arab+) clones. Plasmid DNA from a D-arab+ clone retransformed fresh Ec BW31M cells to D-arab+; these cells produced both ArDH catalytic activity and a 31-kDa protein recognized by antibodies to native Ct ArDH. The plasmid contained an 846-bp open reading frame (ORF) that encoded a deduced protein of 282 amino acids (aa) (30,748 Da). Four partial aa sequences from Ct ArDH were present in the deduced aa sequence, thus verifying that Ct ARD had been cloned. Ct ArDH was 95% identical to ArDH from Candida albicans (Ca), 85% identical to a xylitol dehydrogenase (XDH) from Pichia stipitis (Ps) and 20-25% identical to many other short-chain dehydrogenases. Ct ArDH, Ca ArDH and Ps XDH were typical short-chain dehydrogenases except that they lacked an N-terminal Gly that is conserved in other members of this family. Thus, these enzymes may represent a subclass of closely-related fungal pentitol dehydrogenases. Large amounts of recombinant ArDH (re-ArDH) were produced in Ec and purified by dye ligand affinity chromatography. The physical and catalytic properties of re-ArDH were similar to those of native Ct ArDH, and re-ArDH and native ArDH performed similarly in an automated enzymatic assay for D-arabinitol in human serum.

Amino Acid Sequence↗
PubMed1994

An automated enzymatic method for measurement of D-arabinitol, a metabolite of pathogenic Candida species.

An automated enzymatic method was developed for the measurement of D-arabinitol in human serum. The assay is based on a novel, highly specific D-arabinitol dehydrogenase from Candida tropicalis. This enzyme catalyzes the oxidation of D-arabinitol to D-ribulose and the concomitant reduction of NAD+ to NADH. The NADH produced is used in a second reaction to reduce p-iodonitrotetrazolium violet (INT) to INT-formazan, which is measured spectrophotometrically. The entire reaction sequence can be performed automatically on a COBAS MIRA-S clinical chemistry analyzer (Roche Diagnostic Systems, Inc., Montclair, N.J.). Replicate analyses of human sera supplemented with D-arabinitol over a concentration range of 0 to 40 microM demonstrated that the pentitol could be measured with an accuracy of +/- 7% and a precision (standard deviation) of +/- 0.4 microM. Serum D-arabinitol measurements correlated with those determined by gas chromatography (r = 0.94). The enzymatic method is unaffected by L-arabinitol, D-mannitol, or other polyols commonly found in human serum. Any of 17 therapeutic drugs potentially present in serum did not significantly influence assay performance. Data illustrating the application of the assay in patients for possible diagnosis of invasive candidiasis and the monitoring of therapeutic intervention are presented. The automated assay described here was developed to facilitate the investigation of D-arabinitol as a serum marker for invasive Candida infections.

Agranulocytosis↗
PubMed1993

Identification, purification, and characterization of a D-arabinitol-specific dehydrogenase from Candida tropicalis.

A novel D-arabinitol (DA) dehydrogenase was identified and purified more than 300-fold from Candida tropicalis. The enzyme is specific for DA and catalyzes the NAD(+)-dependent oxidation at carbon 4 to yield D-ribulose. Purification was accomplished by a combination of protamine sulfate and ammonium sulfate precipitation and dye ligand chromatography on a reactive yellow 86 column. The apparent Km of the enzyme for DA ([NAD+] = 2.2 mM) is 39.8 mM. The apparent Km for NAD+ ([DA] = 384 mM) is 0.12 mM. The pH-optimum for the enzymatic oxidation of DA is approximately 10. Cofactor stereospecificity studies demonstrate that the enzyme catalyzes transfer of the 4(S) hydrogen of NADH with D-ribulose as substrate. The polyol substrate specificity of the present DA dehydrogenase makes the enzyme potentially useful for the development of a simple and specific method for the measurement of DA, a metabolite of pathogenic Candida spp. which has been described as a marker for disseminated candidiasis.

Candida↗
PubMed1991

Immunoglobulin variable-region-like domains of diverse sequence within the major histocompatibility complex of the chicken.

The highly polymorphic B-G antigens are considered to be part of the major histocompatibility complex (MHC) of the chicken, the B system of histocompatibility, because they are encoded in a family of genes tightly linked with the genes encoding MHC class I and class II antigens. To better understand these unusual MHC antigens, full-length B-G cDNA clones were isolated from B21 embryonic erythroid cell cDNA library, restriction-mapped, and sequenced. Five transcript types were identified. Analysis of the deduced amino acid sequences suggests that the B-G polypeptides are composed of single extracellular domains that resemble immunoglobulin domains of the variable-region (V) type, single membrane-spanning domains typical of integral membrane proteins, and long cytoplasmic tails. Sequence diversity among the five transcript types was found in all domains, notably including the B-G immunoglobulin V-like domains. The cytoplasmic tails of the B-G antigens are made up entirely of units of seven amino acid residues (heptads) that are typical of an alpha-helical coiled-coil conformation. The heptads vary in number and sequence between the different transcripts. The presence within B-G polypeptides of polymorphic immunoglobulin V-like domains warrants further investigations to determine the degree and nature of variability within this domain in these unusual MHC antigens.

Amino Acid Sequence↗
PubMed1991

A DNA sequence for the discrimination of Neisseria gonorrhoeae from other Neisseria species.

A 350 base pair Neisseria gonorrhoeae DNA restriction fragment was cloned after subtractive hybridization to Neisseria meningitidis DNA. This restriction fragment hybridized to 105 out of 106 N. gonorrhoeae strains tested. While three N. meningitidis strains did not hybridize to this probe, Neisseria mucosa DNA exhibited cross-hybridization. This particular clone was used to screen a N. gonorrhoeae genomic DNA library. A positive 2.4 kilobase pair clone was shown by DNA sequencing to contain two long open reading frames. One open reading frame did not hybridize to N. mucosa and other Neisseria species, while it retained specificity for the original 105 N gonorrhoeae strains. This open reading frame also showed significant homology to cytosine DNA methyltransferases.

Amino Acid Sequence↗
PubMed1988

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex.

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a lambda gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, lambda bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations lambda bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the lambda bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of lambda bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.

Animals↗
PubMed1988

Direct demonstration of critical amino acid residues required for cytotoxic T-lymphocyte allorecognition of H-2 class I antigens.

To identify critical amino acid residues recognized by alloreactive cytolytic T lymphocytes (CTL) generated between H-2Kb and H-2Kbm1, we have derived a series of cloned L-cell lines expressing the following mutant H-2Kb class I genes. Cell line L-KbTyr-Tyr expresses a mutant gene in which positions 155-156 of the Kb molecule have been changed from Arg-Leu to Tyr-Tyr, leaving position 152 unchanged. Cell line L-KbAla expresses the reciprocal mutant gene that has position 152 of the Kb molecule mutated from glutamic acid to alanine, leaving positions 155-156 unchanged. Electrophoretic mobilities of the mutant Kb molecules reflect only those changes predicted by the mutations. Mutant-specific (anti-Kbm1) and native-specific (anti-Kb) CTL lyse L-KbTyr-Tyr and L-KbAla target cells equally well. Unlabeled target inhibition of lysis revealed a pattern of recognition and inhibition that suggests that the amino acid differences between Kbm1 and Kb create at least two discrete determinants that can be recognized by different populations of CTL. The results suggest that these determinants consist, at least in part, of a linear amino acid sequence from which critical amino acid residues can be identified.

Amino Acid Sequence↗
PubMed1986

Liver-specific expression of a Qa-encoded class I gene is associated with DNA hypomethylation.

DNA methylation of two murine major histocompatibility complex (H-2) class I genes was examined in hybridizations to MspI and HpaII chromosomal DNA restriction digests. Q10, which exhibits liver-specific expression, and H-2Kb, a transplantation antigen gene, were examined in liver, spleen, thymus, and cell-line DNAs. Unmethylated Q10 gene sequences were detected only in the liver, whereas the H-2Kb gene was unmethylated in all tissues examined.

Animals↗
PubMed1985

Evidence that polymorphism in the murine major histocompatibility complex may be generated by the assortment of subgene sequences.

The high degree of polymorphism found among the class I genes of the murine major histocompatibility complex (H-2) has led to the postulation that specific genetic mechanisms are responsible for their diversity. These same genetic mechanisms are probably responsible for the high spontaneous mutation frequency seen in H-2 alleles. The bml mutation of the H-2Kb gene has been shown to be 7 base pair changes over a 13 base pair region that result in three amino acid substitutions in the C1 domain of the protein product. The clustering of base-pair changes has suggested that the bm1 mutation resulted from a recombinational event analogous to gene conversion between the H-2Kb gene and a "donor" gene sequence. A 23-base oligonucleotide complementary to the bm1 mutant sequences was synthesized and used to probe genomic DNA restriction digests of the parental H-2b haplotype as well as other H-2 haplotypes. Our results indicate that a potential donor gene sequence is present in the genomes of all of the five mouse strains studied. Of eight tissues that were tested by blot-hybridization analysis, the potential donor gene sequences are transcribed only in the liver. Models for the generation of polymorphism among the H-2 class I genes via subgene rearrangements are proposed.

Animals↗
PubMed1984

Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression.

The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli. Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation). This work describes the identification of the region of the araC promoter that interacts with the cAMP receptor protein to mediate catabolite repression. A 3-base-pair deletion centered 60 base pairs from the transcriptional initiation site results in a mutant araC promoter that, in the absence of araC protein, reduces transcriptional activity when compared with the wild-type promoter and is unresponsive to various concentrations of intracellular cAMP in vivo. The same deletion results in a lowered affinity of the araC promoter for cAMP receptor protein in vitro. However, this lowered affinity for the mutant araC promoter does not result in substantial reduction of intracellular araC protein because autoregulation of the araC gene dominates catabolite repression. The 3-base-pair deletion in the cAMP receptor protein binding site of the araC promoter does not affect catabolite repression of the adjacent araBAD operon. The implications of these results on current models for expression of the araBAD operon and the araC gene are discussed.

Arabinose↗
PubMed1984

Functional limits of the araIc promoter suggest an additional regulatory site for araBAD expression.

The araBAD promoter is defined, in part, by two types of cis-acting constitutive mutations, araIc at position -35 and araXc at position -10. Subcloning experiments demonstrated that the araIc and araIcXc promoters require DNA sequence information out to position -53 to -56 for maximum constitutive expression. This is 8 to 10 base pairs more DNA than is generally thought to be necessary for RNA polymerase interaction. The -53 to -56 region is required for glucose repression, suggesting that an additional factor interacts in this region and is necessary for maximum expression.

Arabinose↗
PubMed1984

Genetic analysis of the functional relationship between colicin E3 and its immunity protein.

Partial deletions in the immunity gene of the colicin E3 operon were used to study possible functions of the immunity protein besides protection against exogenous colicin. Nuclease BAL-31 was used to create a series of carboxyl-terminal deletions of the immunity gene. Mutants displaying lowered immunity against exogenous colicin were found, and six that had reduced but detectable levels of immunity were chosen for further analysis. DNA sequence analysis of the deletions showed that all six terminated within the last five codons of the immunity gene. The wild-type immunity gene was replaced by each of the six mutated immunity genes in a plasmid containing an otherwise functional colicin E3 operon. Transformants containing the resulting plasmids produced smaller colonies on solid medium and grew more slowly in liquid culture than transformants carrying the wild-type colicin and immunity genes. This result suggested that immunity protein was required to protect the cell against endogenous colicin E3. This idea was confirmed in experiments in which the colicin E3 and immunity genes were independently cloned on two compatible plasmid vectors.

Amino Acid Sequence↗