Comparative analyses of multi-species sequences from targeted genomic regions.

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

Temporally unstable recurrence of earthquakes due to breaks in fractal scaling.

Observed sequences of large earthquakes are not consistent in either recurrence time or energy release; long-term prediction has been impossible even in areas, such as Parkfield, with well-defined recurrence intervals. The seismic gap hypothesis, which predicts characteristic earthquakes in areas of the circum-Pacific belt that have not produced recent great earthquakes, has also failed to predict the observed clustering of high-energy events. Models in which fractal scaling is broken at high magnitude predict that characteristic events and recurrence behavior will be unstable in time. The central predictions of these models are supported by recent observations at Landers and Big Bear in California.

Queuine, a modified base incorporated posttranscriptionally into eukaryotic transfer RNA: wide distribution in nature.

Queuine, a modified base found in transfer RNA, appears to be a new dietary factor because (i) previous studies have shown that mice require it for the expression of queuine-containing transfer RNA's but apparently do not synthesize it, and (ii) significant amounts of free queuine are present in common plant and animal food products.

Formation of an adenine-thymine photoadduct in the deoxydinucleoside monophosphate d(TpA) and in DNA.

A photoadduct is formed between the adenine (A) and thymine (T) bases of the deoxydinucleoside monophosphate d(TpA) when it is irradiated at 254 nanometers in aqueous solution. Treatment of the photoadduct with acid converts it specifically into a fluorescent hydrolysis product, C7H7N3O, incorporating the position-8 carbon of adenine and the methyl group of thymine. Isolation of the fluorescent hydrolysis product from acid hydrolyzates of oligo- and polydeoxyribonucleotides has shown that the photoadduct is formed by ultraviolet irradiation of d(pTpA), d(TpApT), d(TpApTpA), poly(dA-dT), and both single- and double-stranded DNA.

The frequency of high-grade intraepithelial neoplasia in anal/perianal warts is higher than previously recognized.

A retrospective review of the prevalence of intraepithelial neoplasia (IN) in surgically removed perianal/anal warts from December 1995 to December 2004 was undertaken in patients referred to the Sexual Health Clinic at Royal Perth Hospital. Data were analysed from 115 men and 38 women, 29 of whom had HIV infection (27 men and two women). Perianal/anal IN within the warts was found in 78% (52% high grade) of men with HIV infection. In men without HIV infection, the overall rate of IN within warts was 33% (20% high grade). The IN rate was 8.3% for HIV-negative women (2.8% high grade). Rates of IN within perianal/anal warts in men with or without HIV infection are higher than previously reported, and suggest the likelihood of a substantial increase in the future incidence of anal cancer. The association between IN and genital warts needs to be further studied.

An open-label phase II pilot study investigating the optimal duration of imiquimod 5% cream for the treatment of external genital warts in women.

Our objective was to determine the optimal duration of treatment with imiquimod for external genital warts over 4, 8, 12 or 16 weeks. A total of 120 women with a history of genital warts for a median of 3-6 months and prior alternative treatments in 73% were evaluated for total clearance rates. There was no statistically significant difference in complete clearance rates after 16-week follow-up across treatment groups: four weeks (40.0%), eight weeks (48.4%), 12 weeks (39.3%) and 16 weeks (51.6%). Imiquimod was well tolerated, and in those treated for four weeks there was a lower incidence of local skin reactions such as erythema and erosion, and no incidences of pain. These preliminary results suggest that a four-week treatment course of imiquimod applied thrice weekly for women with external genital warts may provide a reasonable approach with comparable efficacy and compliance, and minimal adverse events, drug costs and clinic visits.

Selective detection of ribose-methylated nucleotides in RNA by a mass spectrometry-based method.

Post-transcriptional methylation of ribose at position O-2' is one of the most common and conserved types of RNA modification. Details of the functional roles of these methylations are far from clear, although in tRNA they are involved at position 34 in regulation of codon recognition and in eukaryotic rRNAs they are required for subunit assembly. Experimental difficulties in the mapping of ribose methylations increase with RNA molecular size and the complexity of mixtures resulting from nuclease digestion. A new and relatively rapid approach based on tandem mass spectrometry is described in which any of four ion reaction pathways occurring in the mass spectrometer can be monitored which are highly specific for the presence of 2'-O -methylribose residues. These pathways emanate from further dissociation of ribose-methylated mononucleotide (Nmp) ions formed in the electrospray ionization region of the mass spectrometer to then form the base, methylribose phosphate or PO(3)(-)anions. The mass spectrometer can be set for detection of generic ribose methylation (Nm) in oligonucleotides, selectively for each of the common methylated nucleo-sides Cm, Gm, Am or Um or for specific cases in which the base or sugar is further modified. By direct combination of mass spectrometry with liquid chromatography the method can be applied to analysis of complex mixtures of oligonucleotides, as for instance from synthetic or in vitro reaction mixtures or from nuclease digests of RNA. An example is given in which the single ribose-methylated nucleoside in Escherichia coli 16S rRNA (1542 nt), N(4),O-2'-dimethylcytidine, is detected in 25 pmol of a RNase T1 digest and localized to the fragment 1402-CCCGp-1405 in a single 45 min analysis.

Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria.

Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn)and tRNA(Lys)were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys)corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met)corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system. tRNA(Ser)GCU corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or G at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system.

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Isolation and characterization of a novel nucleoside, 7-beta-D-ribofuranosylhypoxanthine, from the urine of a chronic myelogenous leukemia patient.

A novel nucleoside, in the amount of 400 micrograms, was isolated from a 24-h collection of urine of a chronic myelogenous leukemia patient. On the basis of ultraviolet, nuclear magnetic resonance, and mass spectrometry and chromatography, its structure was established to be 7-beta-D-ribofuranosylhypoxanthine. The ultraviolet and mass spectral data and the thin layer chromatographic mobilities of the natural material were identical to those of a synthetic sample. High performance liquid chromatographic retention times and the coinjection high performance liquid chromatography of the natural material with the synthetic samples of the alpha and beta-anomers of 7-ribofuranosylhypoxanthines further confirmed the identity of the isolated material as 7-beta-D-ribofuranosylhypoxanthine.

Reduced misreading of asparagine codons by Escherichia coli tRNALys with hypomodified derivatives of 5-methylaminomethyl-2-thiouridine in the wobble position.

It has been suggested that modified nucleosides of the xm5(s2)U(m)34-type restrict the wobble capacity of the base, and that their function is to prevent misreading in the third position of the codon in mixed codon family boxes that encode two different amino acids. In this study in Escherichia coli, the misreading in vivo of asparagine codons in bacteriophage MS2 mRNA by different hypomodified derivatives of tRNALys, normally containing 5-methylaminomethyl-2-thiouridine (mnm5s2U34) in the wobble position, has been analysed. Contrary to what would be predicted from the general hypothesis for the function of mnm5s2U, it was found that the misreading of asparagine codons by tRNALys was greatly reduced in the mnmA (formerly asuE or trmU) and mnmE (formerly trmE) mutants which contain the hypomodified mnm5U34 and s2U34, respectively, instead of the fully modified mnm5s2U34. In addition, it was found that these hypomodified tRNAs were efficiently charged with lysine in vivo, under the growth conditions employed. The latter result is at variance with results obtained in vitro. The results are discussed in relation to the postulated function for modified nucleosides of the xm5s2U type.

Ultraviolet mutagenesis and its repair in an Escherichia coli strain containing a nonsense codon.

Ultraviolet mutagenesis and its repair were studied mainly in WU36-10-89, a uvr(-) strain of Escherichia coli containing a UAG mutation in a gene for leucine biosynthesis. Following ultraviolet (UV) irradiation revertants appearing with or without direct photoreactivation (PR) were classified according to the presence and type of suppressor they contained. We find UV mutation production to be quite specific. An analysis of revertants produced by UV indicates they are formed mainly from GC --> AT and that the miscoding is due to a cytosine residue at the site of mutation in a cytosine-thymine (CT) dimer. We propose that the dimer serves as template during some aspects of repair replication and at the time of replication the C in the dimer directs the insertion of A in the complementary strand. We also note that C --> A and T -->G changes caused by a CT dimer occur much less frequently.

Applications of mass spectrometry to the characterization of oligonucleotides and nucleic acids.

Mass spectrometry-based techniques continue to undergo active development for applications to nucleic acids, fueled by methods based on electrospray and matrix-assisted laser desorption ionization. In the past two years, notable advances have occurred in multiple interrelated areas, including sequencing techniques for oligonucleotides, approaches to mixture analysis, microscale sample handling and targeted DNA assays, and improvements in instrumentation for greater sensitivity and mass resolution.

Characterization of oligonucleotides and nucleic acids by mass spectrometry.

The continued refinement of two recent methods for producing gas-phase ions, electrospray ionization and matrix-assisted laser desorption ionization, has resulted in new techniques for the rapid characterization of oligonucleotides by mass spectrometry. Using commercially available instruments, molecular mass measurements at the 20-mer level, with errors less than 2 Da, can now be made routinely in less than 15 min. Progress has also been achieved in the development of mass spectrometry for rapid sequencing of oligonucleotides smaller than 25 residues.

Disagreement in high-grade/low-grade intraepithelial neoplasia and high-risk/low-risk HPV infection: clinical implications for anal cancer precursor lesions in HIV-positive and HIV-negative MSM.

Anal condylomata are common in HIV-positive individuals and among men who have sex with men (MSM). Generally attributable to infection by low-risk human papillomaviruses (HPVs), condylomata are considered benign low-grade squamous intraepithelial lesions (SILs). However, anal condylomata have occasionally been linked to high-grade SIL and to oncogenic, high-risk HPVs. Here we describe the range of intraepithelial lesions and of the associated HPVs in heterosexual men and women and MSM. Perianal and anal condylomata were collected from 243 patients (56 heterosexual women, 61 heterosexual men and 126 MSM, including 41 HIV-positive MSM). We assessed lesion histology and HPV genotype. Prevalence estimates and Poisson models were used. Irrespective of HIV infection status, MSM showed a higher proportion of condylomata as high-grade SILs compared to heterosexual men/women. High-grade SILs were also more prevalent in anal than in perianal lesions in all patient groups. HIV-positive MSM exhibited increased prevalence ratio (4.6; 95% confidence interval 2.1-10.0) of perianal low-grade SILs containing only high-risk HPVs compared to HIV-negative MSM. In addition, more than 64% of anal SILs with a high-grade component, regardless of HIV infection, were exclusively associated with low-risk HPVs. In anal condylomata, both high-grade and low-grade SILs can be associated with high-risk and/or low-risk HPVs. Particularly, low-grade perianal SILs associated with high-risk HPVs were common in HIV-positive MSM, while presence of only low-risk HPVs in high-grade SILs were common in both MSM groups. Our findings sound a note of caution for the common clinical practice for the treatment of anal condylomata as benign lesions in MSM and HIV-positive patients.

Implications of a functional large ribosomal RNA with only three modified nucleotides.

The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucleotides could be important structural or functional elements in the catalytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in vitro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose methyltransferase that specifically modifies a universally conserved nucleotide, G2270, in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S). Since the loss of this modification in yeast mitochondrial 21S rRNA severely affects the assembly of 54S subunits, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required for the assembly of 50S subunits. Gm could be a critical structural determinant for the correct folding of the rRNA, the binding of one or more ribosomal proteins, or the interaction of the rRNA with tRNA. Previous work has shown that the mitochondrial large rRNAs are minimally modified relative to the E coli and eukaryotic cytoplasmic rRNAs. By direct chemical analysis using combined high performance liquid chromatography-mass spectrometry, the modification status of the yeast mitochondrial rRNAs was reexamined, revealing the presence of Gm, Um and pseudouridine (psi) in 21S rRNA. The Um was mapped to nucleotide 2791, which corresponds to the ribose methylated and universally conserved U2552 in E coli 23S rRNA, and the psi has been recently mapped to position 2819, which corresponds to psi 2580 in E coli 23S rRNA. The retention of Um and psi nucleotides in the peptidyl transferase center of the otherwise minimally modified mitochondrial rRNAs suggests that these modifications, like Gm2270, might be essential for ribosome assembly or function or both.

Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet).

Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.

Identification of 12-keto-5,8,10-heptadecatrienoic acid as an arachidonic acid metabolite produced by human HL-60 leukemia cells.

An unusual cyclooxygenase-derived metabolite of arachidonic acid has been shown to be produced by N,N-dimethylformamide (DMF)-induced, terminally differentiated human HL-60 promyelocytic leukemia cells and to a much lesser extent by untreated cells. Biochemical evidence in conjunction with gas chromatography/mass spectrometry and liquid chromatography/thermospray mass spectrometry analyses indicates that the product is 12-keto-5,8,10-heptadecatrienoic acid (KHT). Both KHT and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) were produced when arachidonic acid was incubated with cell lysates obtained from differentiated HL-60 granulocytes. Indomethacin and the thromboxane synthetase inhibitor UK-38485 inhibited the production of both metabolites, whereas ethacrynic acid inhibited only the production of KHT. In 100,000 g supernatant fractions, obtained from either untreated or differentiated cells, KHT was produced when HHT was used as substrate. The addition of exogenous NAD, but not NADP, to incubations caused a significant increase in the production of KHT coincident with a decrease in the level of HHT. These data suggest that, in both differentiated and undifferentiated HL-60 cells, an NAD-dependent enzyme, apparently 15-prostaglandin dehydrogenase (15-PGDH), is expressed and catalyzes the conversion of HHT to KHT. In differentiated HL-60 cells, this metabolite is produced from arachidonic acid through a multi-enzymatic process involving the activities of cyclooxygenase, thromboxane synthetase and 15-PGDH. The production of KHT from arachidonic acid in undifferentiated HL-60 cells is probably limited, therefore, by the virtual absence of cyclooxygenase activity in these cells.

Collision-induced dissociation of uracil and its derivatives.

The collision-induced dissociation of protonated uracil has been studied by tandem mass spectrometry using models extensively labeled with stable isotopes, and derivatives of the kinds found in nucleic acids. Following collisional activation at 30 eV translational energy, protonated uracil dissociates through two principal pathways which do not occur in electron ionization mass spectra: (1) elimination of NH3 almost entirely from N-3, followed by loss of CO from C-4, 0(4); (2) loss of H2O, equally from 0(2) and 0(4). Elimination of HNCO, also the principal dissociation process from odd-electron molecular ions, proceeds primarily by loss of N-3, C-Z, O(2) and 10% from N-l, C-Z, 0(2). Several secondary dissociation products are formed with quantitative site specificity of skeletal atoms: C,HO+ (4-C0, C-5, C-6); H2CN+ (N-l, C-6); C2H2+ (N-l, C-5, C-6). First-step dissociation reactions are interpreted in terms of pyrimidine ring opening at likely sites of protonation after collisional activation of MH+. Collision-induced dissociation mass spectra of uracils with structural themes common to nucleic acids (methylation, replacement of 0 by S, C-5 substitution) follow analogous reaction paths which permit assignment of sites of substitution, and exhibit ion abundance changes attributed to differences in substituent basicity and electron density.

Determination of oligonucleotide composition from mass spectrometrically measured molecular weight.

Extensive calculations for molecular mass versus subunit composition have been made for oligonucleotides from RNA and DNA to determine the extent to which base compositions might be derived from mass spectrometrically determined molecular weights. In the absence of compositional constraints (e.g., any numbers of A, U, G, C), measurement of molecular weight leads to only modest restrictions in allowable number of base compositions; however, if the compositional value for any one residue is known, such as from selective chemical modification or enzymatic cleavage, the number of allowable base compositions becomes unexpectedly low. For example, hydrolysis of RNA by ribonuclease T1 produces oligonucleotides for which G=1, for which all base compositions can be uniquely specified up to the 14-mer level, solely by measurement of mass to within ±0,01%. The effects of methylation, phosphorylation state of nucleotide termini, and knowledge of chain length on the determination of subunit composition are discussed.