Biomedical subjects

H A Avila

Publications and source records attributed to H A Avila.

5 recordsLinked to original sources

PubMed1998

U-insertion/deletion Edited Sequence Database.

Uridine insertion/deletion RNA editing is a post-transcriptional RNA modification occurring in the mitochondria of kinetoplastid protozoa. The U-insertion/deletion Edited Sequence Database is a compilation of mitochondrial genes and edited mRNAs from five kinetoplastid species. It contains separate files with the DNA, mRNA (both unedited and edited) and predicted protein sequences, as well as alignments of the Leishmania tarentolae and Trypanosoma brucei protein sequences from edited and unedited genes. The sequence files are in GCG format. A 'map' sequence file showing the location of U-deletions, U-insertions and the translated amino acid sequences is also provided for each gene. Genomic maps for each species are also provided with clickable genes, including maxicircle-encoded gRNAs. Sets of aligned nuclear rRNA sequences from kinetoplastid protozoa are also provided, which were used for phylogenetic reconstructions in an analysis of the origin of RNA editing. The database is available through the World Wide Web as an HTML document at the URLhttp://www.lifesci.ucla.edu/RNA/trypanosome/ database.html

Animals↗

PubMed1995

Organization and complexity of minicircle-encoded guide RNAs in Trypanosoma cruzi.

The previously observed extensive sequence heterogeneity of the kinetoplast minicircle DNA in Trypanosoma cruzi, both intra- and interstrain, has raised the question as to how the minicircle DNA in this species can have any guide RNA (gRNA)-coding capacity at all, because there do not appear to be any variable-region sequences conserved between different strains. To address this question, we obtained the complete edited sequence of maxicircle unidentified reading frame 4 mRNA and identified 25 cognate gRNAs from gRNA libraries constructed from two clonal strains of T. cruzi--Sylvio X10/CL1 and CAN III/CL1. Libraries of PCR-amplified minicircle-variable regions were also constructed for both strains. A single gene for each gRNA was identified in the same polarity within specific minicircle-variable regions from both strains, 60-100 nt downstream from the conserved 12mer sequence. GTP-capped total gRNA from one strain failed to cross-hybridize with minicircle DNA from the other strain. The explanation for this proved to be the number of polymorphisms, mainly transitions, within the homologous gRNAs in the two strains. In most cases, these transitions did not destroy the edited mRNA/gRNA base pairing, as a result of the allowed G-U wobble base pairing. The sequences of the variable regions containing homologous gRNAs in the two strains probably derived from an ancestral sequence, and each has accumulated sufficient polymorphisms so as not to allow hybridization. Within a strain, multiple redundant gRNAs were identified that encode identical editing information but have different sequences.

Amino Acid Sequence↗

PubMed1994

Evolution of RNA editing in kinetoplastid protozoa.

The editing of RNA in trypanosomatid mitochondria involves the insertion and occasional deletion of uridine residues within coding regions of maxicircle messenger RNA transcripts. The extent to which the transcripts of homologous genes undergo editing differs in different species. In some, entire genes are edited (pan-editing), whereas in others, editing is limited to the 5' termini of editing domains (5' editing). Here we investigate which type of editing is ancestral and which is derived, by analysing RNA editing in the different lineages, using a kinetoplastid phylogeny reconstructed from nuclear small subunit ribosomal RNA sequences. We conclude that the ancestral cryptogenes were pan-edited, and we hypothesize that the 5'-edited homologues were generated by several independent events from partially edited RNAs, in which case editing may be a more primitive mechanism than previously thought.

Adenosine Triphosphatases↗

PubMed1993

Detection of Trypanosoma cruzi in blood specimens of chronic chagasic patients by polymerase chain reaction amplification of kinetoplast minicircle DNA: comparison with serology and xenodiagnosis.

A panel of 114 blood samples from chronic chagasic patients and nonchagasic patients was screened for Trypanosoma cruzi by xenodiagnostic, serologic, and polymerase chain reaction (PCR) amplification tests. Blood samples were preserved in a guanidine-EDTA buffer, and total blood DNA was isolated after chemical nuclease cleavage with 1,10-phenanthroline-copper ion and used as a template for PCR amplification of the conserved and variable regions of T. cruzi minicircle molecules. The PCR products were screened by Southern blot hybridization with a digoxigenin-labeled oligonucleotide probe specific for the conserved region of the minicircle. The method showed a sensitivity of 100% compared with the serologic test. In addition, all of the serology-positive, xenodiagnosis-negative samples were positive by PCR. This demonstrates that PCR amplification of T. cruzi kinetoplast minicircle DNA could replace xenodiagnosis for evaluation of parasitemia in chronic chagasic patients and could serve as a complement for serologic testing in the screening of blood bank donors.

Adolescent↗

PubMed1991

Polymerase chain reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA isolated from whole blood lysates: diagnosis of chronic Chagas' disease.

A 6 M guanidine-HCl/0.2 M EDTA solution was used to lyse and store whole blood specimens. DNA stored in guanidine-EDTA-blood (GEB) lysate was found to be undegraded after incubation at 37 degrees C for 1 month, suggesting that this represents an appropriate reagent for transport of blood samples from the field to a laboratory for analysis. Trypanosoma cruzi kinetoplast DNA in GEB lysate can be cleaved using the chemical nuclease, 1,10-phenanthroline-copper ion (OP-Cu2+). This procedure liberates linearized minicircle molecules from network catenation, distributing them throughout the lysate, and allowing a small aliquot of the original lysate to be analyzed by PCR amplification. This increases the sensitivity of the method dramatically for the detection of small numbers of trypanosomes in a large volume of blood. DNAs isolated from aliquots of T. cruzi-positive GEB lysates were polymerase chain reaction (PCR)-amplified with 3 sets of T. cruzi-specific kDNA minicircle primers, yielding the 83-bp and 122-bp conserved region fragments and the 330-bp variable region fragments. The PCR products were analyzed by gel electrophoresis and/or hybridization. Results indicate that a single T. cruzi cell in 20 ml of blood can be detected by this method. Blood samples from several chronic chagasic patients were tested. Amplification of T. cruzi kDNA minicircle sequences was obtained in al cases, even when xenodiagnosis was negative. This PCR-based test should prove useful as a replacement or complement for xenodiagnosis or serology in clinical and epidemiological studies of chronic Chagas' disease.

Animals↗