Solar System Objects Observed in the SDSS Commissioning Data

We discuss measurements of the properties of about 10,000 asteroids detected in 500 deg2 of sky in the Sloan Digital Sky Survey (SDSS) commissioning data. The moving objects are detected in the magnitude range 14 < r < 21.5, with a baseline of 5 minutes. Extensive tests show that the sample is at least 98% complete, with the contamination rate of less than 3%. We find that the size distribution of asteroids resembles a broken power-law, independent of the heliocentric distance: D^{-2.3} for 0.4 km < D < 5 km, and D^{-4} for 5 km < D < 40 km. As a consequence of this break, the number of asteroids with r < 21.5 is ten times smaller than predicted by extrapolating the power-law relation observed for brighter asteroids (r < 18). The observed counts imply that there are about 530,000 objects with D>1 km in the asteroid belt, or about four times less than previous estimates. The distribution of main belt asteroids in the 4-dimensional SDSS color space is bimodal, and the two groups can be associated with S (rocky) and C (carbonaceous) asteroids. A strong bimodality is also seen in the heliocentric distribution of asteroids and suggests the existence of two distinct belts: the inner rocky belt, about 1 AU wide (FWHM) and centered at R~2.8 AU, and the outer carbonaceous belt, about 0.5 AU wide and centered at R~3.2 AU. The colors of Hungarias, Mars crossers, and near-Earth objects are more similar to the C-type than to S-type asteroids, suggesting that they originate in the outer belt. (abridged).Comment: 89 pages, 31 figures, submitted to A

Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair

Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th-5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER

Nasal cathelicidin is expressed in early life and is increased during mild, but not severe respiratory syncytial virus infection

Respiratory syncytial virus is the major cause of acute lower respiratory tract infections in young children, causing extensive mortality and morbidity globally, with limited therapeutic or preventative options. Cathelicidins are innate immune antimicrobial host defence peptides and have antiviral activity against RSV. However, upper respiratory tract cathelicidin expression and the relationship with host and environment factors in early life, are unknown. Infant cohorts were analysed to characterise early life nasal cathelicidin levels, revealing low expression levels in the first week of life, with increased levels at 9 months which are comparable to 2-year-olds and healthy adults. No impact of prematurity on nasal cathelicidin expression was observed, nor were there effects of sex or birth mode, however, nasal cathelicidin expression was lower in the first week-of-life in winter births. Nasal cathelicidin levels were positively associated with specific inflammatory markers and demonstrated to be associated with microbial community composition. Importantly, levels of nasal cathelicidin expression were elevated in infants with mild RSV infection, but, in contrast, were not upregulated in infants hospitalised with severe RSV infection. These data suggest important relationships between nasal cathelicidin, upper airway microbiota, inflammation, and immunity against RSV infection, with interventional potential

NER cleavage reaction products generated using various DNA-RNA-DNA hybrid substrates.

<p><b>I</b>; Cartoon of the synthetic substrates used in the <i>in vitro</i> assays, with the sites of incision and expected product size indicated, along with the DNA sequence containing rNMP(s) and mismatched nucleotides. <b>II</b>; The 50-mer duplexes (10 nM) in which the modified strand was 5′ end-labeled (indicated by *), were incubated with the NER proteins at concentrations of 40 nM (UvrA), 200 nM (UvrB), and 100 nM (UvrC) for 60 min at 55°C in the presence of 1 mM ATP. UvrABC-dependent incision on the fluorescein adducted 50-bp duplex (fT) was used as a control NER activity of the purified proteins (panel A). The DNA duplexes (panel B) as well as DNA-RNA-DNA hybrids either containing a single rAMP (panels C), two consecutive rAMPs (panel D), or five rNMPs (panels E) were assayed. The reaction products were separated under denaturing conditions by 15% polyacrylamide gel electrophoresis (PAGE). The efficiency of UvrABC-dependent incision was determined as a percentage of the radioactivity in the incised products relative to the total signal of the substrate (% inc.). The data below the gels are mean values calculated from at least two independent experiments. The DNA sequence containing rNMP(s) is shown alongside the gels where DNA and RNA are represented by uppercase and lowercase letters, respectively. Orange arrows indicate the cleavage sites. The red bracket indicates spontaneous ribonucleotide cleavage. UDS, refers to the undamaged strand, and DS, is damage/ribonucleotide-containing strand. The <i>in vitro</i> assays reveal that the NER proteins incise the DNA backbone 8 base pairs 5′ of ribonucleotides and that the reactions are stimulated by base mispairs.</p

RNase HII cleavage reaction products generated using various DNA-RNA-DNA hybrid substrates.

<p><b>I</b>; Cartoon of the synthetic substrates used in the <i>in vitro</i> assays, with the sites of incision and expected product size indicated, along with the DNA sequence containing rNMP(s) and mismatched nucleotides. <b>II</b>; The 50-mer duplexes (10 nM) in which the modified strand was 5′ end-labeled (indicated by *), were incubated with Rnase HII for 60 min at 37°C. The DNA duplexes (panel A) as well as DNA-RNA-DNA hybrids containing either single rAMP (panel B), two consecutive rAMPs (panel C), or five rNMPs (panel D) were assayed. The reaction products were separated under denaturing conditions by 15% polyacrylamide gel electrophoresis (PAGE). The efficiency of RNase HII-dependent incision was determined as a percentage of the radioactivity in the incised products relative to the total signal of the substrate (% inc.). The data below the gels are mean values calculated from at least two independent experiments. The DNA sequence containing rNMP(s) is shown alongside the gels where DNA and RNA are represented by uppercase and lowercase letters, respectively. Orange arrows indicate the cleavage sites. The <i>in vitro</i> assays confirm that <i>E.coli</i> RNase HII nicks the DNA backbone 5′ of ribonucleotides embedded in DNA and shows that the efficiency of the reaction is largely unaffected by base-mispairs.</p

Various DNA repair pathways compete, cooperate, or substitute for each other in order to sanitize the <i>E.coli</i> chromosome from mispairs, uracils and incorporated rNMPs.

<p>The cartoon helps to explain why spontaneous mutagenesis induced by wild-type pol V (A) differs from Y11A_UmuC-dependent mutagenesis (B) and illustrates the respective roles of MMR (red box), RER^A (stands for RNase HI-initiated ribonucleotide excision repair and is indicated in yellow), RER^B (RNase HII-initiated ribonucleotide excision repair, indicated in blue), NER (green), and BER (grey) (The competing pathways are indicated by boxes with gradient colors). Misincorporated nucleotides are shown in red (where ts are transitions, tv are transversions), correctly paired ribonucleotides are indicated in blue. For simplicity, all the transversions are shown refractory to MMR and NER while in reality they could be repaired by both pathways although less efficiently than transitions. Both wild-type and mutant pol V make frequent base-substitution errors. The transition mutations are rapidly repaired by MMR, while any errant ribonucleotides (correctly-paired or mispaired) are also efficiently removed by RER^B. Ung-dependent BER only operates on dU, not rU, incorporated into the DNA and therefore has no role in RER. In contrast, NER is able to remove rNMPs misincorporated by either wild-type or mutant pol V. Since <i>umuC</i>_Y11A is able to incorporate multiple consecutive rNMPs into DNA, RER involving RNase HI is limited to strains expressing the pol V variant. RER^B is normally required for highly efficient removal of errant ribonucleotides, however, in its absence the role of NER and RNase HI becomes apparent. The fact that the level of spontaneous mutagenesis in strains expressing <i>umuC</i>_Y11A with an “unlocked” sugar steric gate is 90% lower than mutagenesis in strains harboring wild-type pol V, implies that numerous errant ribonucleotides are very efficiently excised by the collaborative actions of rNMP-specific repair pathways which concomitantly remove mispaired dNMPs positioned within the repair patch (such as for example two D^tvs in the panel B). In the absence of RNase HI, RNase HII and NER proteins, the majority of the misincorporated rNMPs remains embedded in the chromosomal DNA. As a result, spontaneous mutagenesis in the Δ<i>rnhA</i> Δ<i>rnhB</i> Δ<i>uvrA</i> strain expressing <i>umuC</i>_Y11A is higher than in the isogenic strain expressing wild-type pol V.</p

NER cleavage reaction products generated using DNA templates with 3′ end-labeled rNMP-containing strands.

<p><b>I</b>; Cartoon of the synthetic substrates used in the <i>in vitro</i> assays, with the sites of incision and expected product size indicated, along with the DNA sequence containing rNMP(s) and mismatched nucleotides. <b>II</b>; The 50-mer duplexes (10 nM) in which the modified strand was 3′ end-labeled (indicated by *), were incubated with the NER proteins at concentrations of 40 nM (UvrA), 200 nM (UvrB), and 100 nM (UvrC) for 60 min at 55°C in the presence of 1 mM ATP. The DNA duplexes DNA-RNA-DNA hybrids either containing a single rAMP (panel A), two consecutive rAMPs (panel B) were assayed. The reaction products were separated under denaturing conditions by 15% polyacrylamide gel electrophoresis (PAGE). The efficiency of UvrABC-dependent incision was determined as a percentage of the radioactivity in the incised (% inc.) products relative to the total signal of the substrate. The data below the gels are mean values calculated from at least two independent experiments. The DNA sequence containing rNMP(s) is shown alongside the gels where DNA and RNA are represented by uppercase and lowercase letters, respectively. The full-sized DNA template (50-mer) and incision products of 19–20 bp are indicated. Orange arrows indicate the cleavage sites. The <i>in vitro</i> assays reveal that the NER proteins incise the DNA backbone 4–5 base pairs 3′ of ribonucleotides and that the efficiency of the reaction is largely unaffected by base-mispairs.</p