ABSTRACT
BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that are processed from hairpin precursor transcripts by Dicer. miRNAs probably inhibit translation of mRNAs via imprecise antisense base-pairing. Small interfering RNAs (siRNAs) are similar in size to miRNAs, but they recognize targets by precise complementarity and elicit RNA-mediated interference (RNAi). We employed cDNA sequencing and comparative genomics to identify additional C. elegans small RNAs with properties similar to miRNAs and siRNAs. RESULTS: We found three broad classes of small RNAs in C. elegans: (1) 21 new miRNA genes (we estimate that C. elegans contains approximately 100 distinct miRNA genes, about 30% of which are conserved in vertebrates; (2), 33 distinct members of a class of tiny noncoding RNA (tncRNA) genes with transcripts that are similar in length to miRNAs (approximately 20-21 nt) and that are in some cases developmentally regulated but are apparently not processed from a miRNA-like hairpin precursor and are not phylogenetically conserved; (3) more than 700 distinct small antisense RNAs, about 20 nt long, that are precisely complementary to protein coding regions of more than 500 different genes and therefore seem to be endogenous siRNAs. CONCLUSIONS: The presence of diverse endogenous siRNAs in normal worms suggests ongoing, genome-wide gene silencing by RNAi. miRNAs and tncRNAs are not predicted to form complete Watson-Crick hybrids with any C. elegans RNA target, and so they are likely to regulate the activity of other genes by non-RNAi mechanisms. These results suggest that diverse modes of small RNA-mediated gene regulation are deployed in normal worms.
Subject(s)
Caenorhabditis elegans/genetics , MicroRNAs/genetics , Animals , Base Sequence , Blotting, Northern/methods , Caenorhabditis elegans/embryology , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/genetics , Cloning, Molecular , DNA, Complementary/genetics , Gene Library , Genes, rRNA/genetics , Genome , MicroRNAs/chemistry , MicroRNAs/classification , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid Hybridization/methods , RNA, Antisense/chemistry , RNA, Antisense/classification , RNA, Antisense/genetics , RNA, Untranslated/chemistry , RNA, Untranslated/classification , RNA, Untranslated/genetics , Sequence Alignment , Sequence Homology, Nucleic AcidABSTRACT
This study compared the slice method of measuring microleakage to the whole-wall method. Forty-eight Class V direct metal restorations were placed in the buccal and lingual surfaces of 24 human molars. Following thermocycling and storage in 2% methylene blue stain for 12 hours, the teeth were sectioned and the restorations removed to expose the intact occlusal and gingival cavity walls. Maximum dye penetration axially was determined along either one or two imaginary slices or over the whole wall. Data were statistically evaluated by ANOVA and Tukey's test. Non-uniform staining occurred with 38 of the 96 walls available for evaluation. The average maximum dye penetration depth of the 38 walls was 0.61 mm and 0.70 mm for the slice method using one slice or two, respectively, and 1.29 mm for the whole-wall method. About half of these walls had leakage depths that were more than twice as great as when measured by the slice methods. All 12 of the 38 walls with no leakage when measured by the slice method, showed leakage at least somewhere along the margin when measured by the whole-wall method. This study shows that the whole-wall method detects significantly more leakage than does the slice method (p < 0.0001) and that using two rather than one slice does not improve the detection of leakage (p < 0.1534). Slicing the tooth restoration interface into only two or three sections may seriously underestimate the degree of leakage.
