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S D Pena

Publications and source records attributed to S D Pena.

At least 73 records · Page 4Linked to original sources
PubMed1995

PCR haplotypes for the human Y chromosome based on alphoid satellite DNA variants and heteroduplex analysis.

We have developed a system for revealing informative and useful haplotypes for the human Y chromosome using PCR. Variant alphoid satellite DNA subunits were amplified and analysed by digestion with HindIII to score a restriction site polymorphism, or on polyacrylamide gels to reveal 13 heteroduplex haplotypes. Heteroduplexes are double-stranded DNA molecules containing mismatches; the haplotype is the combination of alleles on the same chromosome. Structural studies showed that the heteroduplexes analysed here were formed from loci at the left (short arm) and right (long arm) edges of the centromeric alphoid array which differed by a 4-bp insertion/deletion and several point mutations. Consequently, many haplotypes may have arisen only once and are useful for evolutionary studies.

Base Sequence↗
PubMed1995

Monoclonal antibody against a 52 K sperm surface protein inhibits sperm-zona pellucida interactions in the rat.

A major regulatory site for species specificity of fertilization in mammals lies at the level of sperm binding to the zona pellucida. This implies a high degree of complementarity between gamete and receptor molecules. These molecules support species-specific interactions between sperm and oocyte that lead to gamete fusion (fertilization). We identified a rat sperm head surface antigen using an IgG1 monoclonal antibody (HD1) against rat sperm of epididymis cauda. By electron microscopy the antigen was shown to be present on the plasma membrane surface of the sperm dorsal head. One- and two-dimensional immunoblotting analysis of sperm proteins demonstrated that HD1 reacted only with a 52 K molecule with a pI ranging from 6.6 to 7.2. The 52 K protein was first detected in situ by indirect immunofluorescence and showed to be underlining few elongated spermatids in testis. However, when the sperm reached the epididymis caput, the antigen was seen to be expressed on the dorsal surface of spermatozoa head. A similar fluorescence reaction was detected on sperms in the epididymis corpus and cauda. The specific spermatozoa-zona pellucida interaction was inhibited in the presence of monoclonal antibody HD1 in a sperm binding assay of in vitro fertilization.

Animals↗
PubMed1995

Intracellular promiscuity in Schistosoma mansoni: nuclear transcribed DNA sequences are part of a mitochondrial minisatellite region.

It has been shown that the mtDNA of the parasitic trematode Schistosoma mansoni is hypervariable in size. We report here that this length variation is due to a large polymorphic minisatellite composed of two types of repeated sequences of 558 bp and 62 bp. Each minisatellite repeat is made up of a large 558-bp component and a variable tandem array of the small 62-bp unit. Of more fundamental interest was the finding that both the 558-bp and 62-bp components have significant homology with a gene, SM750, previously identified in the nuclear genome of S. mansoni. The small 62-bp unit is identical to the nuclear polymorphic repeat element, which is apparently spread throughout the nuclear genome and is abundant among transcripts, in addition to being present in five tandem copies in SM750. The presence, in the S. mansoni mtDNA, of fragments of genes that are present in and transcribed from the nuclear genome raises the question of the origin of these sequences. The arrangement and the variability that the mtDNA minisatellite embodies were explored as an identity test for S. mansoni based on the use of PCR for tallying the relative abundance of the several repeat numbers of the tandem arrays of the 62-bp unit within the minisatellite structure.

Animals↗
PubMed1995

Identification of new Schistosoma mansoni genes by the EST strategy using a directional cDNA library.

A directional size-selected cDNA library constructed from Schistosoma mansoni (Sm) adult worm RNA was used for the generation of expressed sequence tags (EST). From one or both ends of 429 distinct cDNA clones 607 EST were obtained. Of these, only 16% were previously known Sm genes. More than 22% of the clones had matches with entries for other organisms in the databases. These new Sm genes constituted a broad range of transcripts distributed among cytoplasmic structural and regulatory proteins, enzymes, membrane, nuclear and secretory proteins, and proteins with other functions. Almost 33% of the clones had no significant database matches and thus potentially represent Sm-specific genes. Among the latter, several clones, as judged by their redundancy in the library, appear to represent abundant transcripts. The data, taken as a whole, more than double the number of Sm genes identified by nucleotide sequencing and indicate the potential value of the adoption of genome sequencing strategies for the rapid increase in knowledge of complex disease-causing organisms.

Animals↗
PubMed1995

Leishmania (Viannia) braziliensis: genetic relationships between strains isolated from different areas of Brazil as revealed by DNA fingerprinting and RAPD.

Leishmania (Viannia) braziliensis isolates from two different geographic areas in Brazil were studied by DNA fingerprinting with the 33.15 multilocal probe and PCR with arbitrary primers (random amplification of polymorphic DNA-RAPD). The genetic distance of strains was measured by band sharing. The results showed that the strains isolated in Minas Gerais, southeastern Brazil, are very different from those isolated in Pará, northern Brazil. Strains from Minas Gerais constituted a relatively homogeneous group, presenting DNA fingerprint patterns with 76% of shared bands and RAPD profiles with 93% of shared bands. In contrast, strains from Pará showed higher genetic variability, with only 17 and 45% of shared bands in DNA fingerprint and RAPD patterns, respectively. This study suggests that genetic differences between L. braziliensis from both areas might have an epidemiological significance.

Animals↗
PubMed1995

DNA diagnosis of human genetic individuality.

DNA studies of the human genome have shown polymorphic variation at thousands of sites, defining an absolute genetic uniqueness for each individual. There are many circumstances in which it may be desirable to diagnose this molecular individuality, as for instance, in criminal investigations or paternity testing. Several techniques can be used for this DNA diagnosis and we can choose among them the one that best suits the specific problem at hand. In this review we describe the main methodologies in current use to investigate human DNA polymorphisms, discussing the best application of each option, as well as their advantages and disadvantages.

Bioethics↗
PubMed1995

Yeast artificial chromosome (YAC)-based genome mapping of Schistosoma mansoni.

Schistosoma mansoni has 7 pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7 x 10(8) bp. We initiated the molecular genetic approach for the detailed characterization and understanding of the evolutionary biology of schistosomes. We have constructed a yeast artificial chromosome (YAC)-library with partially digested parasite genomic DNA, and the chromosome location of each insert was detected by fluorescent in situ hybridization (FISH). The library contains > 2283 clones with an average insert size of 358 kb, which represents a 2.6-fold coverage of the genome (> 7.2 x 10(8) bp). 100 randomly selected YAC clones were localized by FISH and found to be distributed widely among all chromosomes. The assembly of 14 YACs distributed almost the whole region of chromosome 3. Generated expressed sequenced tags (ESTs) derived from a unidirectional cDNA library were also used for further characterization of the YAC inserts. These results indicate that an extensive contig assembly of the entire chromosomes and a reasonably detailed gene map should be feasible in the near future.

Animals↗
PubMed1994

Sequence-specific "gene signatures" can be obtained by PCR with single specific primers at low stringency.

Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA.

Animals↗
PubMed1994

Allelic associations of two polymorphic microsatellites in intron 40 of the human von Willebrand factor gene.

At intron 40 of the von Willebrand factor (vWF) gene, two GATA-repeat polymorphic sites exist that are physically separated by 212 bp. At the first site (vWF1 locus), seven segregating repeat alleles were observed in a Brazilian Caucasian population, and at the second (vWF2 locus) there were eight alleles, detected through PCR amplifications of this DNA region. Haplotype analysis of individuals revealed 36 different haplotypes in a sample of 338 chromosomes examined. Allele frequencies between generations and gender at each locus were not significantly different, and the genotype frequencies were consistent with their Hardy-Weinberg expectations. Linkage disequilibrium between loci is highly significant with positive allele size association; that is, large alleles at the loci tend to occur together, and so do the small alleles. Variability at each locus appeared to have arisen in a stepwise fashion, suggesting replication slippage as a possible mechanism of production of new alleles. However, we observed an increased number of haplotypes, in contrast with the predictions of a stepwise production of variation in the entire region, suggesting some form of "cooperative" changes between loci that could be due to either gene conversion, or a common control mechanism of production of new variation at these repeat polymorphism sites. The high degree of polymorphism (gene diversity values of 72% and 78% at vWF1 and vWF2, respectively, and of 93% at the haplotype level) makes these markers informative for paternity testing, genetic counseling, and individual-identification purposes.

Adult↗
PubMed1994

Kinetoplast DNA from Trypanosoma rangeli contains two distinct classes of minicircles with different size and molecular organization.

Trypanosomatids are characterized by the presence of kinetoplast DNA (kDNA), a peculiar form of mitochondrial DNA that consists of several thousand minicircles and a few dozen maxicircles catenated in a network. Within a species, the minicircles are known to differ in nucleotide sequence, but are homogeneous in size and always cross-hybridize. In all species of trypanosomatids, kDNA minicircles have at least one copy of a conserved 100-200 nucleotide region containing an almost invariant 'universal' 12-mer sequence (5'-GGGGTTGGTGTA-3'). We here report that Trypanosoma rangeli, a non-pathogenic parasite of man, contains two distinct classes of kDNA, minicircles called KP1 and KP2, which differ in size and molecular organization. Both were cloned and sequenced in both directions. KP2 was 1587 bases along and contained two copies of the conserved region as direct repeats 180 degrees apart. In contrast, KP1 had 1764 bases and showed a single conserved region. Moreover, KP1 differed further from KP2 and from most other previously sequenced trypanosomatid minicircles by containing a nucleotide substitution (5'-GGGGTTAGTGTA-3') in the 12-mer universal sequence tag. Polymerase chain reaction and hybridization studies suggest that the sequence of KP1 is very conserved in several other T. rangeli strains from Honduras, Colombia and Venezuela. It thus could provide a good target for the molecular diagnosis of infection with this parasite.

Animals↗
PubMed1994

Paternity testing in the DNA era.

Paternity can be efficiently and effectively tested using multi-allelic variable number of tandem repeat (VNTR) polymorphisms. This can be achieved by using multilocus DNA fingerprinting probes or batteries of single-locus minisatellite probes, or by PCR testing of sets of minisatellite and microsatellite loci. Although some problems of statistics and population genetics remain to be overcome, the informativeness of these hypervariable loci is so high that paternity disputes can be resolved with certainty in virtually every case.

Chromosome Mapping↗
PubMed1993

Paternity testing with the F10 multilocus DNA fingerprinting probe.

Empirical analysis of 200 paternity cases by multilocus DNA fingerprinting with the F10 probe showed that it was capable of distinguishing fathers from non-fathers in every case. The average exclusion probability was 0.99998. A very effective discrimination parameter was the proportion of non-maternal (test) bands which cannot be detected in the alleged father (unassignable bands) among all test bands. Values below 0.2 were seen in true fathers while in all cases of non-fathers the values were above 0.35. Minisatellite mutations occurred at a rate of 0.004 per band per child. The distribution of band-sharing among first degree relatives and unrelated individuals showed only a small overlap. Thus, band-sharing of the F10 fingerprints should provide a useful statistic for testing genetic relationships in deficiency cases.

Base Sequence↗
PubMed1993

Testing deficiency paternity cases with a Y-linked tetranucleotide repeat polymorphism.

Because a son's genotype at a Y-linked locus uniquely specifies his father's genotype at that locus irrespective of the maternal contribution, Y-linked polymorphisms show increased exclusion power over autosomal polymorphisms in paternity cases involving a male child. This advantage is even more obvious when the alleged father is deceased or otherwise unavailable for testing. In this situation, any individual connected by patrilineage to the alleged father may be tested in his place. The usefulness of the Y-linked tetranucleotide repeat locus Y-27H39 in deficiency cases was evaluated in a set of 41 families that had a deceased alleged father and that had been tested at GENE/MG with multilocal and unilocal DNA fingerprinting probes. In sixteen of these cases the proband (child tested) was male and there were male relatives. In the thirteen cases in which paternity was included, Y-27H39 would contribute significantly to the paternity index. In one of three cases in which there was exclusion by fingerprinting probes there was also exclusion by Y-27H39. Thus, Y-27H39 is useful in deficiency paternity cases and will be specially valuable in situations where autosomal polymorphisms have limited power, such as when there is only one male living relative of the deceased father. However, our experience is that Y-linked probes can only be applied in approximately 40% of cases.

Base Sequence↗