The monogonont rotifer Brachionus plicatilis is an emerging model system for a diverse array of questions in limnological ecosystem dynamics, the evolution of sexual recombination, cryptic speciation, and the phylogeny of basal metazoans. We sequenced the complete mitochondrial genome of B. plicatilis sensu strictu NH1L and found that it is composed of 2 circular chromosomes, designated mtDNA-I (11,153 bp) and mtDNA-II (12,672 bp). Hybridization to DNA isolated from mitochondria demonstrated that mtDNA-I is present at 4 times the copy number of mtDNA-II. The only nucleotide similarity between the 2 chromosomes is a 4.9-kbp region of 99.5% identity including a transfer RNA (tRNA) gene and an extensive noncoding region that contains putative D-loop and control sequence. The mtDNA-I chromosome encodes 4 proteins (ATP6, COB, NAD1, and NAD2), 13 tRNAs, and the large and small subunit ribosomal RNAs; mtDNA-II encodes 8 proteins (COX1-3, NAD3-6, and NAD4L) and 9 tRNAs. Gene order is not conserved between B. plicatilis and its closest relative with a sequenced mitochondrial genome, the acanthocephalan Leptorhynchoides thecatus, or other sequenced mitochondrial genomes. Polymerase chain reaction assays and Southern hybridization to DNA from 18 strains of Brachionus suggest that the 2-chromosome structure has been stable for millions of years. The novel organization of the B. plicatilis mitochondrial genome into 2 nearly equal chromosomes of 4-fold different copy number may provide insight into the evolution of metazoan mitochondria and the phylogenetics of rotifers and other basal animal phyla.

BackgroundRotifers are among the most common non-arthropod animals and are the most experimentally tractable members of the basal assemblage of metazoan phyla known as Gnathifera. The monogonont rotifer Brachionus plicatilis is a developing model system for ecotoxicology, aquatic ecology, cryptic speciation, and the evolution of sex, and is an important food source for finfish aquaculture. However, basic knowledge of the genome and transcriptome of any rotifer species has been lacking.Methodology/Principal FindingsWe generated and partially sequenced a cDNA library from B. plicatilis and constructed a database of over 2300 expressed sequence tags corresponding to more than 450 transcripts. About 20% of the transcripts had no significant similarity to database sequences by BLAST; most of these contained open reading frames of significant length but few had recognized Pfam motifs. Sixteen transcripts accounted for 25% of the ESTs; four of these had no significant similarity to BLAST or Pfam databases. Putative up- and downstream untranslated regions are relatively short and AT rich. In contrast to bdelloid rotifers, there was no evidence of a conserved trans-spliced leader sequence among the transcripts and most genes were single-copy.Conclusions/SignificanceDespite the small size of this EST project it revealed several important features of the rotifer transcriptome and of individual monogonont genes. Because there is little genomic data for Gnathifera, the transcripts we found with no known function may represent genes that are species-, class-, phylum- or even superphylum-specific; the fact that some are among the most highly expressed indicates their importance. The absence of trans-spliced leader exons in this monogonont species contrasts with their abundance in bdelloid rotifers and indicates that the presence of this phenomenon can vary at the subphylum level. Our EST database provides a relatively large quantity of transcript-level data for B. plicatilis, and more generally of rotifers and other gnathiferan phyla, and can be browsed and searched at gmod.mbl.edu.

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.

1. The use of everted sacs of the small intestine as an enzyme source for studying the metabolism of xenobiotics by cytochrome P450 (P450, CYP) enzymes and UDP-glucuronosyltransferases has been investigated. 2. Most of the drug oxidation activities for testosterone, bufuralol, ethoxyresorufin and 7-ethoxycoumarin resided in the upper part of the everted sacs and in intestinal microsomes. Testosterone 6 beta-hydroxylase activities in the everted sacs were about two times higher than those in the intestinal microsomes. By freezing and thawing treatment, the testosterone 6 beta- and 16 alpha-hydroxylase activities of the everted sacs were considerably decreased, and those of the intestinal microsomes were abolished. 3. Microsomal testosterone 6 beta-hydroxylation, bufuralol 1'-hydroxylation, and pentoxyresorufin and ethoxyresorufin O-dealkylation were inhibited by ketoconazole, quinine, metyrapone and alpha-naphthoflavone respectively. Immunoreactive proteins using anti-CYP2B1 and anti-CYP3A2 antibodies were detected in the upper and middle parts of the rat small intestine. 4. Except for morphine 3-glucuronidation, glucuronidation activities in intestinal microsomes or everted sacs were not dependent on the intestinal region. The lower part of the everted sacs exhibited about 10 times higher morphine-3-glucuronidation activities compared with those of the upper part. The glucuronidation activities of 4-nitrophenol in the everted sacs were 10 times higher than those in microsomes. 5. These results demonstrated that the upper part of rat small intestine serves as the major site for intestinal P450-mediated first-pass metabolism. CYP3A enzymes in rat intestinal microsomes may not be stable but probably play an important role in drug oxidations. The high activity of glucuronidation in the rat small intestine should also be considered in terms of drug metabolism.