Summary MicroRNAs (miRNAs) and small‐interfering RNAs (siRNAs) have emerged as important regulators of gene expression in higher eukaryotes. Recent studies indicate that genomes in higher plants encode lineage‐specific and species‐specific miRNAs in addition to the well‐conserved miRNAs. Leguminous plants are grown throughout the world for food and forage production. To date the lack of genomic sequence data has prevented systematic examination of small RNAs in leguminous plants. Medicago truncatula, a diploid plant with a near‐completely sequenced genome has recently emerged as an important model legume. We sequenced a small RNA library generated from M. truncatula to identify not only conserved miRNAs but also novel small RNAs, if any. Eight novel small RNAs were identified, of which four (miR1507, miR2118, miR2119 and miR2199) are annotated as legume‐specific miRNAs because these are conserved in related legumes. Three novel transcripts encoding TIR‐NBS‐LRR proteins are validated as targets for one of the novel miRNA, miR2118. Small RNA sequence analysis coupled with the small RNA blot analysis, confirmed the expression of around 20 conserved miRNA families in M. truncatula. Fifteen transcripts have been validated as targets for conserved miRNAs. We also characterized Tas3‐siRNA biogenesis in M. truncatula and validated three auxin response factor (ARF) transcripts that are targeted by tasiRNAs. These findings indicate that M. truncatula and possibly other related legumes have complex mechanisms of gene regulation involving specific and common small RNAs operating post‐transcriptionally.

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In this investigation of radical formation and reaction in gamma- irradiated DNA and model compounds, we report the conversion of the guanine cation radical (one-electron oxidized guanine, G(.+)) to the C1' sugar radical and another sugar radical at the C3' or C4' position (designated C3'(.)/C4'(.)) by visible and UV photolysis. Electron spin resonance (ESR) spectroscopic investigations were performed on salmon testes DNA as well as 5'-dGMP, 3'-dGMP, 2'-deoxyguanosine and other nucleosides/nucleotides as model systems. DNA samples (25- 150 mg/ml D(2)O) were prepared with Tl(3+) or Fe(CN)(3-)(6) as electron scavengers. Upon gamma irradiation of such samples at 77 K, the electron-gain path in the DNA is strongly suppressed and predominantly G(.+) is found; after UV or visible photolysis, the fraction of the C1' sugar radical increases with a concomitant reduction in the fraction of G(.+). In model systems, 3'- dGMP(+.) and 5'-dGMP(+.) were produced by attack of Cl(.-)(2) on the parent nucleotide in 7 M LiCl glass. Subsequent visible photolysis of the 3'-dGMP(+.) (77 K) results predominantly in formation of C1'(.) whereas photolysis of 5'-dGMP(+.) results predominantly in formation of C3'(.)/C4'(.). We propose that sugar radical formation is a result of delocalization of the hole in the electronically excited base cation radical into the sugar ring, followed by deprotonation at specific sites on the sugar.

Electron spin resonance (ESR) studies of radicals formed by radiation-induced multiple one-electron oxidations of guanine moieties in DNA are reported in this work. Annealing of gamma-irradiated DNA from 77 to 235 K results in the hydration of one electron oxidized guanine (G*+) to form the 8-hydroxy-7,8-dihydroguanin-7-yl-radical (*GOH) having one beta-proton coupling of 17-28 G and an anisotropic nitrogen coupling, A(parallel), of approximately 20 G, A(perpendicular) = 0 with g(parallel) = 2.0026 and g(perpendicular) = 2.0037. Further annealing to 258 K results in the formation of a sharp singlet at g = 2.0048 with line-width of 5.3 G that is identified as the 8-oxo-7,8-dihydroguanine one-electron-oxidized radical (8-oxo-G*+). This species is formed via two one-electron oxidations of *GOH. These two one-electron oxidation steps leading to the formation of 8-oxo-G*+ from *GOH in DNA, are in accordance with the expected ease of oxidation of *GOH and 8-oxo-G. The incorporation of oxygen from water in G*+ leading to *GOH and to 8-oxo-G*+ is verified by ESR studies employing 17O isotopically enriched water, which provide unambiguous evidence for the formation of both radicals. ESR analysis of irradiated-DNA in the presence of the electron scavenger, Tl3+, demonstrates that the cationic pathway leads to the formation of the 8-oxo-G*+. In irradiated DNA-Tl3+ samples, Tl3+ captures electrons. Tl2+ thus produced is a strong oxidant (2.2 V), which is metastable at 77 K and is observed to increase the formation of G*+ and subsequently of 8-oxo-G*+ upon annealing. We find that in the absence of the electron scavenger the yield of 8-oxo-G*+ is substantially reduced as a result of electron recombinations with G*+ and possible reaction with *GOH.