Examinando por Autor "Villegas, J."

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    A putative RNA editing from U to C in a mouse mitochondrial transcript
    (Oxford University Press, 2002-05) Villegas, J.; Müller, I.; Arredondo, J.; Pinto, R.; Burzio, L.O.
    Recently, we isolated and characterized a new mouse mitochondrial RNA molecule containing the mitochondrial 16S RNA plus 121 nt joined to the 5′ end of the RNA. This fragment arises from the L strand of the same gene and we have named this transcript chimeric RNA. At position 121 of the RNA there is a C, which, according to the sequence of the mitochondrial 16S RNA gene, should be a U. We hypothesized that this RNA is synthesized having a U at position 121, which is later substituted to a C by a putative editing reaction. Based on the presence of sites for the restriction endonucleases RsaI and Fnu4HI around position 121, both forms of the RNA were detected in mouse tissues. To confirm the presence of the non-edited and putative edited RNA, a fragment containing the first 154 nt of the RNA was amplified by RT–PCR and cloned. The substitution of U for C was demonstrated by sequencing these clones. In vitro transcription experiments demonstrated that the substitution of U for C is not due to artifact of amplification or cloning. Moreover, in mitochondria from testis only the non-edited form was found. This, together with other experimental evidence, demonstrated that the base substitution was not due to polymorphism of the mitochondrial 16S RNA gene. This is the first demonstration of a substitution reaction from U to C in a mammalian mitochondrial transcript.
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    Determination of the differential expression of mitochondrial long non-coding RNAs as a noninvasive diagnosis of bladder cancer
    (2013) Rivas, A.; Burzio, V.; Landerer, E.; Borgna, V.; Gatica, S.; Ávila, R.; Lópe, C.; Villota, C.; De La Fuente, R.; Echenique, J.; Burzio, L.; Villegas, J.
    Bladder cancer is a significant cause of morbidity and mortality with a high recurrence rate. Early detection of bladder cancer is essential in order to remove the tumor, to preserve the organ and to avoid metastasis. The aim of this study was to analyze the differential expression of mitochondrial non-coding RNAs (sense and antisense) in cells isolated from voided urine of patients with bladder cancer as a noninvasive diagnostic assay. Methods. The differential expression of the sense (SncmtRNA) and the antisense (ASncmtRNAs) transcripts in cells isolated from voided urine was determined by fluorescent in situ hybridization. The test uses a multiprobe mixture labeled with different fluorophores and takes about 1 hour to complete. We examined the expression of these transcripts in cells isolated from urine of 24 patients with bladder cancer and from 15 healthy donors. Results: This study indicates that the SncmtRNA and the ASncmtRNAs are stable in cells present in urine. The test reveals that the expression pattern of the mitochondrial transcripts can discriminate between normal and tumor cells. The analysis of 24 urine samples from patients with bladder cancer revealed expression of the SncmtRNA and down-regulation of the ASncmtRNAs. Exfoliated cells recovered from the urine of healthy donors do not express these mitochondrial transcripts. This is the first report showing that the differential expression of these mitochondrial transcripts can detect tumor cells in the urine of patients with low and high grade bladder cancer. Conclusion: This pilot study indicates that fluorescent in situ hybridization of cells from urine of patients with different grades of bladder cancer confirmed the tumor origin of these cells. Samples from the 24 patients with bladder cancer contain cells that express the SncmtRNA and down-regulate the ASncmtRNAs. In contrast, the hybridization of the few exfoliated cells recovered from healthy donors revealed no expression of these mitochondrial transcripts. This assay can be explored as a non-invasive diagnostic tool for bladder cancer.
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    Localization of the 16S mitochondrial rRNA in the nucleus of mammalian spermatogenic cells
    (Oxford University Press, 2002-11) Villegas, J.; Araya, P.; Bustos-Obregon, E.; Burzio, L.
    Amplification of RNA from human sperm heads yielded a fragment of 435 bp that shares 100% identity with a central region of the 16S mitochondrial rRNA. The nuclear localization in the sperm of the mitochondrial RNA was confirmed by in-situ hybridization. These results, together with the localization of the 16S mitochondrial rRNA in mouse sperm, are the first demonstration that the organelle transcript is a normal component of the mammalian gamete. The possibility that the nuclear mitochondrial RNA arises from nuclear transcription of a mitochondrial pseudogene was ruled out. To determine when during spermatogenesis the mitochondrial RNA is localized in the nucleus, in-situ hybridization of mouse and human testis was carried out. The nuclei of spermatogonia, spermatocytes and round and elongated spermatids were all positively stained. In human spermatocytes, the nuclear staining pattern was fibrillar, suggesting an association of the mitochondrial transcript with the meiotic chromosomes. These results indicate that early in spermatogenesis and before the onset of meiosis, the 16S mitochondrial rRNA is localized in the nucleus of spermatogenic cells, suggesting a process of translocation of the transcript from the mitochondria.