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D M Iovannisci

Publications and source records attributed to D M Iovannisci.

13 recordsLinked to original sources
PubMed1997

Introduction to PCR/OLA/SCS, a multiplex DNA test, and its application to cystic fibrosis.

The field of medical, molecular diagnostics has grown rapidly over the last few years, becoming increasingly informative to both clinician and patient. As genes associated with specific diseases have been discovered and sequenced, many genotype-phenotype relationships have been defined. For those genetic diseases with associated, defined, gene mutations, sophisticated DNA diagnostic tests are being developed. As an example, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, is associated with Cystic Fibrosis (CF). We have developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF. Test design in the field of molecular diagnostics must consider such characteristics as specificity, sensitivity, ease and speed of protocol, multiplex capacity, and cost. PCR/OLA/SCS addresses these requirements. Polymerase Chain Reaction (PCR) is widely used in both diagnostics and research. We have combined well established PCR technology with Oligonucleotide Ligation Assay (OLA) and Sequence-Coded Separation (SCS), two relatively new technologies.

Alleles↗
PubMed1995

Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations.

Isolation of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), provided a basis for analyzing its molecular pathology and resulted in the identification of > 400 mutations associated with disease. Except for the delta F508 mutation, no other single mutation accounts for > 5% of CF chromosomes in most populations, and most mutation frequencies are < 1%. A strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was used to detect 30 mutations, distributed throughout ten exons and seven introns of the CFTR gene, that together account for > 96% of CF mutant chromosomes worldwide. Mutations were detected by competitive oligonucleotide probe ligation to detect normal and/or mutant genotypes in one reaction. Three probes (one common and two allelic probes) were needed for analysis of each mutation. Probes hybridized to target DNA were joined by a thermostable ligase if there were no mismatches at their junctions; temperature cycling resulted in a linear increase in product. Common probes were labeled with fluorochromes, and allelic probes each had different lengths. Ligation products were analyzed electrophoretically on a fluorescent DNA sequencer. The results show that combined PCR and probe ligation amplification rapidly and reliably screen for CF homozygotes and carriers.

Alleles↗
PubMed1994

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

Autoanalysis↗
PubMed1993

Ligation amplification and fluorescence detection of Mycobacterium tuberculosis DNA.

Current methods for the identification of Mycobacterium tuberculosis are dependent upon culture of the bacteria and are necessarily lengthy due to the slow growth of this agent. The development of DNA probe technology offers rapid, accurate and cost effective alternatives for the identification of such fastidious organisms. A technique for detecting specific DNA sequences, known as oligonucleotide ligation assay (OLA) involves the ligation of two adjacent oligonucleotides annealed to target DNA, and has been previously described. Amplification of the target sequences can be accomplished by including complementary pairs of oligonucleotides and a thermal stable ligase in a reaction which cycles between annealing/ligation and denaturing temperatures. Using a cloned portion of an insertion sequence, IS6110, which has been reported to be specific for M. tuberculosis complex as target DNA, we demonstrate the ligation dependent amplification of a 40 base pair region of plasmid bearing IS6110. By employing oligonucleotides which are each labelled with a different fluorescent dye, the reaction can be followed by fluorescence detection on an Applied Biosystems model 373A DNA sequencer. Using this approach, we have optimized conditions for the detection of 100 target molecules in a mixture containing 4 micrograms of unrelated DNA. Since the insertion sequence is repeated on average 12-14 times in the genome of M. tuberculosis, this corresponds to a theoretical detection level of 7-8 organisms. Completion of this entire assay can be accomplished in less than 8 h and serves as a basis for further studies in the development of a rapid clinical diagnostic test for tuberculosis.

Base Sequence↗
PubMed1989

Structural alterations of chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of the mini-exon array.

We have utilized pulsed field electrophoresis to characterize several karyotypic alterations in Leishmania major. Promastigotes of the LT252 line contain three small chromosomes, of 300, 350 and 385 kb. Quantitative densitometry of ethidium bromide-stained gels suggest that these chromosomes are present in equal levels (2:2:2). Two derivatives of this line, one appearing spontaneously (LT252 delta) and one obtained following selection with methotrexate (11-MTXR20), exhibit altered levels of these chromosomes, in the ratio of 2:1:3, respectively. The variant pattern in both lines is due to an increase in size of chromosome 2, yielding a new chromosome similar in size to chromosome 3. The enlarged chromosome 2 of the LT252 delta line is a result of amplification of the mini-exon gene array normally located on this chromosome, which increases from about 93 to 150 copies of the 0.44-kb mini-exon tandem repeat, as shown by quantitative hybridization and sizing of the mini-exon array. In contrast, the increased size of chromosome 2 within the methotrexate-resistant mutant 11-MTXR20 is not due to mini-exon amplification. In both variant lines, there are equal levels of the wild-type and enlarged chromosome 2, and the wild-type chromosome 2 is now present at 50% of the level of chromosome 1. These and other data suggest that Leishmania is diploid for chromosomes bearing housekeeping genes such as the mini-exon locus.

Animals↗
PubMed1986

Adenine phosphoribosyltransferase-deficient Leishmania donovani.

Mutant promastigotes of Leishmania donovani deficient in adenine phosphoribosyltransferase (APRTase) have been isolated in medium containing 4-aminopyrazolopyrimidine. The generation of APRTase-deficient mutants occurred in two discrete steps. In the first step, clones were isolated with 50% of wildtype levels of APRTase activity. These cells were reselected and colonies totally deficient in APRTase were isolated. Partially and totally APRTase-deficient cells exhibited intermediate and complete resistance to cytotoxic adenine analogs, respectively. Nevertheless, wildtype and mutant cells could salvage adenine and utilize adenine as a purine source equally efficiently, suggesting that the adenine deaminase-HGPRTase pathway plays an important role in promastigote adenine metabolism. Kinetic and thermal inactivation studies of purified APRTase and isoelectric focusing of crude extracts from wildtype and partially APRTase-deficient cells suggested that the latter cells possessed wildtype APRTase activity at half the amount found in wildtype parental cells. These data suggest that Leishmania donovani possess two copies of the APRTase structural gene and that these organisms might be diploid for the APRTase locus.

Adenine↗
PubMed1984

Genetic analysis of adenine metabolism in Leishmania donovani promastigotes. Evidence for diploidy at the adenine phosphoribosyltransferase locus.

Mutant promastigotes of Leishmania donovani deficient in adenine phosphoribosyltransferase (AP-Rib transferase) have been isolated in medium containing 4-aminopyrazolopyrimidine. The generation of AP-Rib transferase-deficient mutants occurred in two discrete steps. In the first step, clones were isolated with 50% of wild-type levels of AP-Rib transferase activity. These cells were reselected, and colonies totally deficient in AP-Rib transferase were isolated. Wild-type and AP-Rib transferase-deficient cells contained equivalent amounts of other enzymes essential to adenine metabolism such as adenine deaminase and hypoxanthine-guanine phosphoribosyltransferase. Partially and totally AP-Rib transferase-deficient cells exhibited intermediate and complete resistance to cytotoxic adenine analogs, respectively. Nevertheless, wild-type and mutant cells could salvage adenine and utilize adenine as a purine source equally efficiently, suggesting that the adenine deaminase-hypoxanthine-guanine phosphoribosyl-transferase pathway plays an important role in promastigote adenine metabolism. Kinetic and thermal inactivation studies of purified AP-Rib transferase and isoelectric focusing of crude extracts from wild-type and partially AP-Rib transferase-deficient cells suggested that the latter cells possessed wild-type AP-Rib transferase activity at half the amount found in wild-type parental cells. These data suggest that L. donovani possesses two copies of the AP-Rib transferase structural gene and that these organisms might be diploid for the AP-Rib transferase locus.

Adenine↗
PubMed1984

Biochemical genetic analysis of formycin B action in Leishmania donovani.

Formycin B is cytotoxic toward Leishmania and is a potential chemotherapeutic agent for leishmaniasis. In order to determine the mechanism of action of formycin B, we have isolated and characterized clonal populations of formycin B-resistant Leishmania donovani. These formycin B-resistant clones are also cross-resistant to formycin A and allopurinol riboside-mediated growth inhibition. Incubation of the formycin B-resistant cells with [3H]formycin B indicates that, unlike wild type cells, the resistant populations cannot accumulate phosphorylated metabolites of exogenous [3H]formycin B. This is due to a defective transport system for formycin B in the resistant cells. However, wild type and mutant cells incorporate [3H]formycin A equally efficiently into [3H]formycin A-containing nucleotides and into RNA. These data suggest that formycin B cytotoxicity in Leishmania is not mediated by its incorporation as the adenosine analog into RNA. A plausible alternative hypothesis is proposed for the mechanism of action of the pyrazolo (4,3-d)pyrimidine C-nucleosides based upon depletion of an essential intracellular metabolite.

Allopurinol↗
PubMed1984

Single cell cloning of Leishmania parasites in purine-defined medium: isolation of drug-resistant variants.

A simple technique for the isolation of Leishmania donovani and Leishmania tropica promastigote clones derived from a single cell involves the use of semi-solid agar. Both species of Leishmania form discrete colonies at high efficiency in completely defined medium lacking serum. Visible colonies appear between eight and fourteen days. Viability of colonies transferred from semi-solid agar to liquid suspension culture is 100%. Using these techniques, we have isolated clonal populations of cells resistant to tubercidin and formycin b.

Agar↗
PubMed1984

Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity.

From a mutagenized population of wildtype Leishmania donovani promastigotes, a clonal cell line, TUBA2, was isolated by virtue of its ability to survive and grow in 20 microM tubercidin (7-deazaadenosine). The TUBA2 clone was also 1000-fold less sensitive than the parental line to growth inhibition by formycin A, another cytotoxic adenosine analog. Parental and mutant cells, however, were equally sensitive to growth inhibition by formycin B, allopurinol riboside, and 6-thioguanosine. Mutant cell extracts, unlike those prepared from wildtype cells, did not phosphorylate radiolabelled adenosine, tubercidin, or formycin A. Intact adenosine kinase-deficient cells did not accumulate exogenous tubercidin or formycin A but incorporated [14C]adenosine at rates 25% of those found for parental cells. The uptake data suggest that adenosine kinase plays an important role in the metabolism of adenosine but indicate alternative metabolic pathways for this nucleoside. The metabolism of adenosine to the nucleotide level in TUBA2 cells appears to be initiated via deribosylation to adenine. Significant amounts of both adenosine hydrolytic and adenosine phosphorylytic activities have been detected in L. donovani promastigotes. Furthermore, L. donovani extracts could slowly catalyze the deamination of formycin A. The isolation and characterization of adenosine kinase-deficient cells has provided considerable insight into the function of the purine pathway in L. donovani.

Adenosine↗
PubMed1984

Genetic analysis of nucleoside transport in Leishmania donovani.

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.

Animals↗
PubMed1983

High efficiency plating method for Leishmania promastigotes in semidefined or completely-defined medium.

A simple technique, developed for the isolation of clones derived from single, promastigote cells of Leishmania donovani and Leishmania tropica, involved the use of semisolid agar. Both species of Leishmania promastigotes formed discrete colonies at high efficiency either in semidefined medium containing 10% fetal calf serum or in completely-defined medium lacking serum. Visible colonies appeared between 8 and 14 days in growth medium containing 10% fetal calf serum. Replacement of the fetal calf serum with bovine serum albumin and Tween-80 increased the time of colony formation by 50% but did not affect the cloning efficiency. Viability of colonies transferred from semisolid agar to liquid suspension culture was 100%.

Agar↗