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A Maddalena

Publications and source records attributed to A Maddalena.

14 recordsLinked to original sources
PubMed1992

Nalidixic acid-resistant V79 cells with reduced DNA topoisomerase II activity and amplification prone phenotype.

Spontaneously nalidixic acid-resistant lines (NAr lines) were selected from a V79 Chinese hamster cell line and phenotypically characterized. NAr lines showed an increased doubling time, a higher number of spontaneous SCE, and more interestingly, decreased DNA topoisomerase II activity. These lines were also cross-resistant to the eukaryotic topoisomerase II inhibitors etoposide and adriamycin, but showed the same level of sensitivity as the parental line to the DNA topoisomerase I inhibitor camptothecin. NAr lines were cross-resistant to other drugs, such as PALA, MTX and MPA, resistance to which has been shown to arise by amplification of the target genes. This last feature, together with enhanced cross-resistance to PALA and MTX when employed simultaneously, suggests that NAr lines have an 'amplification prone' phenotype. From these results the decreased activity of topoisomerase II seems to be involved in the generation of amplified sequences possibly by affecting recombinational events underlying gene amplification.

Animals↗
PubMed1992

Reliable gender screening for human preimplantation embryos, using multiple DNA target-sequences.

Dependable methods were developed for preimplantation sexing of human IVF embryos, for use in clinical settings where prospective parents are at high risk for transmission of X-linked diseases. Using single cultured cells and blastomeres from human embryos as model systems, a multiplex protocol was developed for rapid analysis via nested polymerase chain reaction (PCR). Reliability was enhanced by co-amplification of conserved amelogenin gene segments from both X and Y chromosomes, as well as Y-linked DYZ1 repetitive elements. Each cell was manually isolated and individually washed to avoid potential contaminants. Multiplex amplification allowed recognition of spurious amplification failures specific to particular amelogenin single-copy targets. The X-linked internal control and multiple Y-linked markers allowed recognition and exclusion of most aberrant samples, thus averting potential misdiagnosis. The optimized single-cell protocol reduced experimental sexing errors to < 2% (1/60), but also revealed potential pitfalls of single-cell analysis. With human triploid embryos, separate sampling of individual blastomeres provided concordant female or male signals. Slight modification adapted the procedure for diagnosis of biopsy material from blastocyst stage embryos, allowing separate analysis of multiple tubes containing multiple cells for improved reliability.

Amelogenin↗
PubMed1992

Molecular diagnosis of genetic disease.

DNA-based testing is becoming possible for an increasing number of hereditary diseases as the responsible genes are mapped to individual chromosomes and then isolated and characterized. The strategy for each test depends on the heterogeneity of mutations commonly causing the disease, the distribution of the mutations in the population and the frequency of new mutations. In many cases, interpretation of the test result requires a comparison with relatives known to carry the abnormal gene. Sickle cell anemia is an example of a recessive mutation with a strong ethnic association and little genetic heterogeneity or new mutation. Cystic fibrosis also has a strong ethnic association and a low frequency of new mutation but greater heterogeneity. Fragile X affects all ethnic groups and often appears sporadic, but all cases have the same type of mutation, and a premutation can be found in all affected families. In the near future, the extreme sensitivity of DNA analysis will allow testing for these and similar diseases to be performed in vitro on fertilized embryos before implantation.

Blotting, Southern↗
PubMed1989

Prenatal diagnosis and heterozygote detection by DNA analysis in ornithine transcarbamylase deficiency.

This report summarizes our experience with DNA analysis using a complementary DNA probe for ornithine transcarbamylase in 24 individuals or families with deficiency of this enzyme. In four cases, including three reported elsewhere, a Taql restriction site alteration directly detected the mutation. In 10 additional cases, only an affected male was available, and results of DNA analysis using the Taql enzyme were normal. In 10 cases, family studies were performed with the use of restriction fragment length polymorphisms. Prenatal diagnostic studies were performed for three informative pregnancies, and two affected male fetuses were identified. Analysis of two restriction fragment length polymorphisms, Mspla and BamHl, was informative in 14 of 19 (74%) known carrier females and in 21 of 35 (60%) females (the total number studied). One female previously predicted to be a noncarrier by protein-loading test was determined to be a carrier by analysis of restriction fragment length polymorphisms. The frequency of Taql site alterations was 4 of 24 families (17%). These data illustrate the importance of DNA analysis, pedigree analysis, and biochemical testing in families with ornithine transcarbamylase deficiency to detect carriers and establish the diagnosis prenatally.

Blotting, Southern↗
PubMed1989

Cytogenetic manifestations associated with the reversion, by gene amplification, at the HGPRT locus in V79 Chinese hamster cells.

Some HGPRT spontaneous revertants were isolated from a mutant line (E2) of V79 Chinese hamster cells and phenotypically characterized. Dot-Blot hybridization with a 32P-labelled HGPRT probe revealed an increase in the number of HGPRT sequences in some of these revertants, suggesting the occurrence of gene amplification. Cytogenetic analysis performed in three of these revertants showed a characteristic abnormally banding region (ABR) on the elongated p arm of the X chromosome. In situ hybridization in one revertant (RHE2) showed that the amplified sequences reside on the p+ arm of the X chromosome in two different localizations. Because of the very probably clonal origin of the revertant, these features indicate that the amplified sequences might rearrange after their integration into the chromosome.

Animals↗
PubMed1988

Characterization of point mutations in the same arginine codon in three unrelated patients with ornithine transcarbamylase deficiency.

Point mutations in the X-linked ornithine transcarbamylase (OTC) gene have been detected at the same Taq I restriction site in 3 of 24 unrelated probands with OTC deficiency. A de novo mutation could be traced in all three families to an individual in a prior generation, confirming independent recurrence. The DNA sequence in the region of the altered Taq I site was determined in the three probands. In two unrelated male probands with neonatal onset of severe OTC deficiency, a guanine (G) to adenine (A) mutation on the sense strand (antisense cytosine [C] to thymine [T]) was found, resulting in glutamine for arginine at amino acid 109 of the mature polypeptide. In the third case, where the proband was a symptomatic female, C to T (sense strand) transition converted residue 109 to a premature stop. These results support the observation that Taq I restriction sites, which contain an internal CG, are particularly susceptible to C to T transition mutation due to deamination of a methylated C in either the sense or antisense strand. The OTC gene seems especially sensitive to C to T transition mutation at arginine codon 109 because either a nonsense mutation or an extremely deleterious missense mutation will result.

Adult↗
PubMed1985

Antigen-specific anti-phosphocholine antibodies: binding site studies.

The present investigation extends our initial evaluation of the evolution of antigen selection mechanisms for antibodies of a "single" specificity. The binding sites of 11 mouse anti-PC antibodies produced in response to the bacterium P. morganii or the nematode A. suum were characterized for both hapten and hapten plus carrier specificity. All of the anti-P. morganii HP belonged to the M603 anti-PC antibody family, whereas all the A. suum HP belonged to the M511 family. Of the eight anti-P. morganii HP, six exhibited a fine specificity profile for PC and choline analogues only slightly different from M603 Id+ HP induced by S. pneumoniae and PC-protein. These six and a seventh HP, whose hapten binding profile was unique, were also unusual in showing strong reactivity for a soluble PC containing extract from P. morganii. All three anti-A. suum-specific HP studied in detail had hapten-binding profiles remarkably similar to each other, a finding that is in contrast to M511 Id+ HP to S. pneumoniae and PC-protein. All three HP also showed evidence for preferential binding activity for A. suum, although this was not as dramatic as that seen with the anti-P. morganii HP. These data support our hypothesis that antigen selection of anti-PC antibodies occurs not so much for PC itself as it does for the carrier (microbial) determinants to which PC is attached.

Animals↗
PubMedDate not supplied

Recent experience in prenatal diagnosis of fragile X.

Our experience with 48 prenatal fragile X cases (35 amniocenteses; 13 chorionic villi samplings) is summarized. Of these 48 cases, 18 consultands were known to be carriers of fragile X. No cytogenetic false negatives or positives were identified but 2 cases were uninterpretable. Cytogenetic recommendations include: 1) Ten or more days recovery time after growth in Chang medium, and 2) use of 3-4 media/inducer systems with duplicate harvests. Direct DNA probe testing will likely be the method of choice for prenatal diagnosis after sufficient data are collected to verify interpretation. Until then, both cytogenetic and direct DNA techniques should be utilized.

Amniocentesis↗