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Biomedical subjects

J Montoya

Publications and source records attributed to J Montoya.

At least 73 records · Page 4Linked to original sources
PubMed1992

Molecular characterization and cloning of sheep mitochondrial DNA.

Mitochondrial DNA from the liver of a single Rasa Aragonesa sheep has been isolated and characterized. The size of the genome, determined by restriction enzyme analysis, was found to be 16.58 kbp. The cleavage sites for the restriction endonucleases BamHI, HindIII, EcoRI, BglII, PvuII, BstEII and PstI were mapped, and the gene organization deduced through heterologous hybridization using different cloned fragments of the rat mitochondrial genome. Fragments representative of the entire sheep genome were cloned in plasmid vectors pGEM3Z and pUN121.

Animals↗
PubMed1992

A simple procedure for recovering the denaturing effect of methylmercury in agarose gel electrophoresis.

A simple and rapid procedure for recovering the denaturing effect of methylmercuric hydroxide in agarose gel electrophoresis is described. The procedure consisted of the treatment of the commercial methylmercuric hydroxide solutions with Amberlite, a mixture of anion- and cation-exchange resins. This treatment greatly improved the resolution of RNA species when fractionated by electrophoresis through agarose-CH3HgOH slab gel.

Animals↗
PubMed1991

Estimation of the chloramphenicol and cycloheximide inhibition of protein synthesis in brain cholinergic synaptosomes.

Cholinergic synaptosomes have been prepared from sheep brain cortex by means of an immunoaffinity method using a specific anti-(Chol I) antiserum. The [14C]leucine incorporation into proteins of this preparation shows a low cycloheximide and a high chloramphenicol sensitivity. This fact suggests that the mitochondrial protein synthesis system is the only one present in this fraction.

Animals↗
PubMed1991

Saturation of the processing of newly synthesized rRNA in isolated brain mitochondria.

Isolated rat brain mitochondria, when incubated in the presence of [alpha-32P]UTP in an appropriate incubation buffer, in which the energy requirements are provided by exogenous ADP in the presence of an oxidizable substrate, are able to support mitochondrial DNA transcription and RNA processing in a way faithfully resembling the in vivo process. Furthermore, we have strikingly found a saturation of the synthesis of mature 16 S and 12 S rRNA under conditions in which their RNA precursor as well as the mature mRNAs continue being synthesized. This suggests that synthesis of mature rRNAs could be regulated at the level of processing of their precursor rather than at the level of transcription.

Adenosine Diphosphate↗
PubMed1991

Analysis of polyadenylated RNA from human heart mitochondria.

The content of the mitochondrial poly(A)-RNA fraction of human heart has been analyzed by electrophoresis through agarose slab gels in the presence of methyl mercury hydroxide and staining with ethidium bromide. It is possible to identify all the heavy strand coded mRNAs in the electrophoretic pattern. This pattern is qualitatively similar to that obtained from the mitochondria of cells cultured in vitro (HeLa cells), although differences in the relative amount of some components have been detected.

Electrophoresis, Agar Gel↗
PubMed1990

Analysis of polyadenylated RNA from brain synaptosomes and mitochondria.

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.

Animals↗
PubMed1990

Development of Mansonella perstans in the surrogate vector Aedes aegypti.

The biology of the human filaria Mansonella perstans has been poorly studied due in part to the lack of experimental animal models in which its life-cycle could be reproduced. In nature Culicodes spp. (and probably simuliids) are the vectors involved in disease transmission. In the present work Aedes aegypti black-eyed strain was experimentally infected with M. perstans microfilariae by intrathoracic inoculation of 8 to 15 parasites contained in 0.4-0.6 microliters of RPMI 1640. Concentration of microfilariae was achieved by means of a ficoll separation technique. A. aegypti were maintained at 26 degrees C and 80% relative humidity with a sugar-water diet, except on day 4 post-infection on which they received an uninfected blood meal. Larval development was slow, taking no less than 4 days to reach the sausage stage, which measured 95-100 microns in length. Molt to the second stage took place on the 6th day; the differentiation into a long muscular and glandular esophagus, and short intestine measuring approximately one fourth of the total larval length were the most significant changes. At the end of this period 4 small but well demarcated tail papillae were observed. The first third-stage larvae appeared at the 8th day post-infection, measuring 650 to 680 microns in length. Beyond the 10th day larvae with an average length of 750 microns were found in the thorax, head, and mouthparts. Four conspicuous tail papillae characteristic of the genus Mansonella were seen in all third-stage larvae.(ABSTRACT TRUNCATED AT 250 WORDS)

Aedes↗
PubMed1989

Mechanism of mRNA binding to bovine mitochondrial ribosomes.

The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs. In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U] shorter than about 10 nucleotides. The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch. Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes. We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure. Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract. Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors. Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria.

Animals↗
PubMed1988

Leishmaniasis in Colombia. I. Studies on the phlebotomine fauna associated with endemic foci in the Pacific Coast region.

Studies on the phlebotomine fauna related to the leishmaniasis endemic foci of the Colombian Pacific Coast were carried out in the municipalities of Tumaco and Buenaventura. In Inguapí del Guadual, Tumaco, Lutzomyia trapidoi and Lu. gomezi were the predominant anthropophilic species; Lu. panamensis and Lu. hartmanni were less frequent. In Bajo Calima, Buenaventura, Lu. trapidoi represented over 94% of the anthropophilic sandflies. Continuous sampling from 1800 to 0600 hours in Inguapí del Guadual demonstrated that Lu. trapidoi bites mainly at dusk and dawn whereas Lu. gomezi remains active throughout the night. In Inguapí del Guadual, promastigotes were found in 0.1% (2/2, 305) of Lu. trapidoi, 0.2% (3/140) of Lu. gomezi, and 0.2% (1/424) of Lu. panamensis samples collected. In Bajo Calima, 1.9% (8/429) of Lu. trapidoi were found to be infected. Leishmania braziliensis panamensis, the most common Leishmania subspecies in the human population of this endemic focus, was isolated from 1 Lu. trapidoi from Inguapí del Guadual. Parasitological and entomological findings suggest that Lu. trapidoi could be the main vector of Leishmania in these areas, although Lu. gomezi and Lu. panamensis were also predominant.

Animals↗
PubMed1984

A new type of EcoRI polymorphism of the human ribosomal DNA repeating unit revealed by analysis of cloned DNA fragments.

Several clones of rDNA have been isolated from an adult human liver DNA Charon 4A library by using cDNA probes synthesized from human 18S and 28S rRNA. The insert of one recombinant Charon 4A clone contained, besides the already known 5.7-kb EcoRI fragment of rDNA, comprising the major portion of the 18S rRNA gene and all the external transcribed spacer (ETS), a previously unidentified EcoRI fragment of rDNA of 8.5 kb in size. DNA transfer hybridization experiments utilizing EcoRI digests of the human DNA used to construct the library and of another human DNA showed the presence of the 8.5-kb EcoRI fragment in a minority of the rDNA repeats on the 5'-end side of the 5.7-kb fragment, thus defining a hitherto unidentified type of EcoRI polymorphism of these repeats.

Adult↗
PubMed1983

The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription units.

A detailed analysis of the mapping and kinetic properties of oligo(dT)-cellulose bound and unbound transcripts synthesized in HeLa cells has indicated that two distinct transcription events take place in the rDNA region. Of these, one appears to start approximately 25 bp upstream of the tRNAPhe gene and to terminate at or near the 3' end of the 16S rRNA gene, being responsible for the synthesis of the bulk of rRNA. The other transcription event starts near the 5' end of the 12S rRNA gene, proceeds beyond the 3' end of the 16S rRNA gene, and results in the synthesis of a polycistronic molecule corresponding to almost the entire H-strand, which is destined to be processed to yield the mRNAs and most of the tRNAs encoded in the heavy strand. The existence of two overlapping transcription units with distinct promoters is probably the basis for the differential regulation of synthesis of the rRNAs and heavy-strand-coded mRNAs.

DNA↗
PubMed1982

Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA.

The initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mitochondrial DNA have been investigated by mapping experiments utilizing in vitro "capped" mitochondrial RNA molecules or nascent RNA chains. Mitochondrial poly(A)-containing RNA molecules were labeled at their 5' ends with [alpha-32P]GTP and guanylyltransferase ("capping" enzyme) and mapped on the mitochondrial genome by DNA transfer hybridization and S1 nuclease protection experiments. A mapping site for the capped 5' ends was found on the H strand very near to the 5' terminus of the 12S rRNA gene, and another site was found on the L strand very near to the 5' terminus of the 7S RNA coding sequence. In parallel experiments, the 5' ends of the nascent chains isolated from mitochondrial DNA transcription complexes were similarly mapped very near to the 5' termini of the 12S rRNA gene and of the 7S RNA coding sequence. The in vitro capped RNA molecules and the nascent chains thus presumably identify the same transcriptional initiation sites on the H strand and the L strand. The occurrence of a second possible initiation site for H-strand transcription 90-110 nucleotides upstream of that described above--i.e., 20-40 nucleotides upstream of the tRNAPhe gene--had been previously indicated by a mapping analysis of the nascent RNA chains and has been confirmed in the present work. The presence of two initiation sites for H-strand transcription can be correlated with other types of evidence that point to two different transcription events leading to the synthesis of a polycistronic molecule corresponding to the almost entire H strand and to the synthesis of the rRNA species.

Chromosome Mapping↗