Since alterations in post-transcriptional events can contribute to the appearance and/or progression of cancer, we investigated whether RNA editing, catalyzed by the ADAR (adenosine deaminases that act on RNA) enzymes, is altered in pediatric astrocytomas. We find a decrease in ADAR2 editing activity that seems to correlate with the grade of malignancy in children. Despite the loss of ADAR2 editing activity in tumor tissues, the high grade astrocytomas do not exhibit alterations in ADAR2 expression when compared with their specific control tissues. However, high expression levels of ADAR1 and ADAR3 were found in tumors when compared with normal tissues dissected in the same area of the brain. We reintroduced either ADAR2 or the inactive version of ADAR2 in three astrocytoma cell lines (U118, A172, U87). The "reverted" editing status is necessary and sufficient for a significant decrease in cell malignant behavior as measured by proliferation, cell cycle, and migration assays. We show that elevated levels of ADAR1, as found in astrocytomas, do indeed interfere with ADAR2 specific editing activity. Furthermore, we show that the endogenous ADAR1 can form heterodimers with ADAR2 in astrocytes.The ADARs 3 (adenosine deaminases that act on RNA) are enzymes responsible for adenosine (A) to inosine (I) conversion in pre-mRNA. This deamination is the most widespread type of RNA editing in higher eukaryotes. Since the translation machinery reads inosine as guanosine (1), the ADARs can modify protein codons and thus modulate protein sequence and function of several gene products. The editing of a specific adenosine is not usually 100% efficient and as a consequence, the ADARs can generate different protein isoforms in the same cell.
A tandemly repeated unit of 6553 bp containing a copy of the four core histone genes H2B, H2A, H3, and H4, and also a 5S rRNA gene, was amplified by PCR from genomic DNA of the isopod crustacean Asellus aquaticus. The linkage between 5S rRNA genes and histone genes has been so far observed in only one other organism, the anostrac crustacean Artemia salina. The gene cluster was cloned and sequenced. The histone genes, in their 3' flanking region, have the interesting feature of possessing two different mRNA termination signals, the stem-loop structure and the AATAAA sequence. A part of the PCR product was used as a probe in FISH experiments to locate the gene cluster on an inter-individually variable number of chromosomes from 6 to 12 per diploid cell, always in a terminal position and never associated with the heterochromatic areas. Fluorescence in situ hybridization (FISH) was also performed on preparations of released chromatin and the reiteration level of the gene cluster was determined as approximately 200-300 copies per haploid genome.
We investigated the 5S ribosomal RNA (rRNA) genes of the isopod crustacean Asellus aquaticus. Using PCR amplification, three different tandemly repeated units containing 5S rDNA were identified. Two of the three sequences were cloned and sequenced. One of them was 1842 bp and presented a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene. This type of linkage had never been observed before. The other repeat consisted of 477 bp and contained only an incomplete 5S rRNA gene lacking the first eight nucleotides and a spacer sequence. The third sequence was 6553 bp long and contained a 5S rRNA gene and the four core histone genes. The PCR products were used as probes in fluorescent in situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus. The possible evolutionary origin of the three repeated units is discussed.
In this investigation we analysed the 5S rRNA genes of the isopod crustacean Proasellus coxalis, 5S rDNA hybridization of digested genomic DNA and amplification by PCR demonstrate that these genes are organized in tandem repeats of 589 bp, 120 of which represent the coding sequence and 469 the spacer sequence. Proasellus coxalis is the first crustacean species in which 5S rRNA genes have been found tandemly arranged without being linked to other repeated genes. The PCR product has been used as a probe in FISH to locate the 5S rRNA genes on two chromosome pairs of the P. coxalis karyotype. Comparison of the 5S rRNA sequence of this species with previously published sequences of six other crustacean species shows the existence of a good correlation between phylogenetic relationships and sequence identity.
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