DNA Sequence Effects on Single Base Deletions Arising during DNA Polymerization in Vitro by Escherichia Coli Klenow Fragment Polymerase

Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent study, were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.     

A structural model for sequence-specific proflavin-DNA interactions during in vitro frameshift mutagenesis.

Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5' pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it. Because the most frequent mutations are opposite Pu in the template sequence 5' Py Pu 3', a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin. The model seeks to rationalize the roles of the 5' pyrimidine and proflavin in facilitating the bypass. Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5' pyrimidine; 4) hydrogen bonding with the phosphate backbone. These models point to the possibility that a number of proflavin-DNA interactions may be involved. In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.     

Effects of proflavin and photoactivated proflavin on the template function of single-stranded DNA

DNA context-specific effects of the association of proflavin, single-stranded DNA and DNA polymerase on DNA polymerization reactions were examined. Frameshift mutations induced by the presence of proflavin during in vitro DNA replication of a single-stranded DNA template by the Klenow fragment of Escherichia coli DNA polymerase I were sequenced. More than 80% of the frameshifts were one base-pair deletions opposite purine bases that were immediately 3' to pyrimidines. Purines (Pu) that were not adjacent to pyrimidines (Py) were not deletion sites. The remaining deletions were opposite template pyrimidines that were also immediately 3' to another pyrimidine. All pyrimidine site deletions occurred in the context 5' PyPyPu 3'. In additional experiments, the site-specific inhibition of processive DNA polymerization by proflavin was examined. A novel inhibition of polymerization was found opposite all pyrimidines in the template when proflavin-template complexes were exposed to ten seconds of white light. This inhibition of polymerization is reversible. Longer photoactivation led to an altered pattern of DNA sequence-specific inhibition that was not reversible. The role of DNA sequence-specific interactions of proflavin with DNA in proflavin mutagenesis is discussed.     

Polymerase-specific differences in the DNA intermediates of frameshift mutagenesis. In vitro synthesis errors of Escherichia coli DNA polymerase I and its large fragment derivative

The sequences of more than 600 frameshift mutations produced as a consequence of in vitro DNA replication on an oligonucleotide-primed, single-stranded DNA template by the Escherichia coli polymerase I enzyme (PolI) or its large fragment derivative (PolLF) were compared. Four categories of mutants were found: (1) single-base deletions, (2) base substitutions, (3) multiple-base deletions and (4) complex frameshift mutations that change both the base sequence and the number of bases in a concerted mutational process. The template sequence 5'-Py-T-G-3', previously identified as a PolLF hotspot for single-base deletions opposite G, is also a hotspot for PolI. A PolI-specific warm spot for single-base deletions was identified. Among base substitutions, transitions were more frequent than transversions. Transversions were mediated by (template)G.G, (template)G.A, and (template)C.T mispairs. Multiple-base deletions were found only after PolI replication. Although each of these deletions can be explained by a misalignment mediated by directly repeated DNA sequences, deletion frequencies were often different for repeats of the same length. Both PolI and PolLF produced many complex frameshift mutants. The new sequences at the mutant sites are exactly complementary to nearby DNA sequences in the newly synthesized DNA strand. In each case, palindromic complementarity could mediate the misalignment needed to initiate the mutational process. The misaligned DNA synthesis accounts for the nucleotide changes at the mutant site and for homology that could direct realignment of the DNA onto the template. Most of the complex mutant sequences could be initiated by either intramolecular misalignments involving fold-back structures in newly synthesized DNA or by strand-switching during strand-displacement synthesis. The striking differences between the specificities of complex frameshift mutations and multiple-base deletions by PolI and PolLF identify the existence of polymerase-specific determinants that influence the frequency and specificity of misalignment-mediated frameshifts and deletions.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

A Structural Model For Sequence-Specific Proflavin-DNA Interactions During In Vitro Frameshift Mutagenesis

Abstract

Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5′ pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it. Because the most frequent mutations are opposite Pu in the template sequence 5′ Py Pu 3′, a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin. The model seeks to rationalize the roles of the 5′ pyrimidine and proflavin in facilitating the bypass. Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5′ pyrimidine; 4) hydrogen bonding with the phosphate backbone. These models point to the possibility that a number of proflavin-DNA interactions may be involved. In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.     

An in vitro approach to identifying specificity determinants of mutagenesis mediated by DNA misalignments

In vitro, misalignments of the newly synthesized (primer) strand during DNA polymerization lead to deletion and/or complex frameshift mutations. In vivo, similar misalignments of repeated and quasipalindromic DNA sequences are predicted to be intermediates of mutagenesis. The mutagenic misalignments are mediated by complementary pairing between the sequence at the 3'-OH end of the newly synthesized DNA strand and sequences in the template or in the newly synthesized DNA. Mutant sequences are produced when the misaligned primers act as substrates for DNA polymerization. The misalignments responsible for detected mutant sequences were compared to similar misalignments that were not implicated in mutagenesis, and all misalignment possibilities were compared to the position of pausing during polymerization by Escherichia coli polymerase I or its Klenow fragment. These comparisons revealed three characteristics of in vitro misalignment specificity. First, the termini produced by pausing are likely to be precursors to mutagenic misalignments. Second, the absence of some potential misalignments from the detected spectrum is explained well by the predicted undetectability of the mutant sequences they produce. Third, factors distinct from pausing and mutant detectability are responsible for differences in the specificity of misalignment mutagenesis mediated by E. coli DNA polymerase I and Klenow polymerase during in vitro synthesis.     

Human DNA polymerases lambda and beta show different efficiencies of translesion DNA synthesis past abasic sites and alternative mechanisms for frameshift generation

Human DNA polymerases (pols) beta and lambda could promote template slippage and generate -1 frameshifts on defined heteropolymeric DNA substrates containing a single abasic site. Kinetic data demonstrated that pol lambda was more efficient than pol beta in catalyzing translesion DNA synthesis past an abasic site, particularly in the presence of low nucleotide concentrations. Moreover, pol lambda was found to generate frameshifts in two ways: first, by using a nucleotide-stabilized primer misalignment mechanism, or second, by promoting primer reannealing using microhomology regions between the terminal primer sequence and the template strand. Our results suggest a molecular mechanism for the observed high in vivo rate of frameshifts generation by pol lambda and highlight the remarkable ability of pol lambda to promote microhomology pairing between two DNA strands, further supporting its proposed role in the nonhomologous end joining process.     

Strand asymmetry of +1 frameshift mutagenesis at a homopolymeric run by DNA polymerase III holoenzyme of Escherichia coli

We have recently shown that single-base frameshifts were predominant among mutations induced within the rpsL target sequence upon oriC plasmid DNA replication in vitro. We found that the occurrence of +1 frameshifts at a run of 6 residues of dA/dT could be increased proportionally by increasing the concentration of dATP present in the in vitro replication. Using single-stranded circular DNA containing either the coding sequence of the rpsL gene or its complementary sequence, the +1 frameshift mutagenesis by DNA polymerase III holoenzyme of Escherichia coli was extensively examined. A(6) --> A(7) frameshifts occurred 30 to 90 times more frequently during DNA synthesis with the noncoding sequence (dT tract) template than with the coding sequence (dA tract). Excess dATP enhanced the occurrence of +1 frameshifts during DNA synthesis with the dT tract template, but no other dNTPs showed such an effect. In the presence of 0.1 mM dATP, the A(6) --> A(7) mutagenesis with the dT tract template was not inhibited by 1.5 mM dCTP, which is complementary to the residue immediately upstream of the dT tract. These results strongly suggested that the A(6) --> A(7) frameshift mutagenesis possesses an asymmetric strand nature and that slippage errors leading to the +1 frameshift are made during chain elongation within the tract rather than by misincorporation of nucleotides opposite residues next to the tract.     

The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis

The frequency and specificity of mutations produced during in vitro DNA synthesis of the lacZ alpha gene in M13mp2 DNA by eucaryotic DNA polymerase-alpha (pol-alpha) and DNA polymerase-gamma (pol-gamma) have been determined. Pol-alpha, purified from five different sources, produces mutations resulting in loss of alpha-complementation at a frequency of 0.8-1.6%/single round of gap-filling DNA synthesis. DNA sequence analysis of 420 independent mutants produced by pol-alpha demonstrates three classes of errors. The majority of mutations result from single base substitutions, while single base frameshifts are detected at a lower but substantial frequency. Large deletions are also observed, with a frequency and specificity suggesting that they too are produced by pol-alpha in vitro. In contrast, pol-gamma is more accurate, producing mutants at a frequency of 0.3-0.5%. The specificity of pol-gamma errors is also different, since more than 90% of the mutants result from single base substitutions, while frameshift errors are not observed at a frequency significantly above background. The pol-gamma mutant spectrum also contains deletion mutations (10 of 179 mutants) presumably resulting from aberrant in vitro synthesis. When considered together with previous results using pol-beta (Kunkel, T. A. (1985) J. Biol. Chem. 260, 5787-5796) the relative accuracy of the three classes of purified vertebrate DNA polymerases for base substitutions, frameshifts, and deletions is in the order gamma greater than alpha greater than beta. These data demonstrate a correlation between the accuracy and processivity of DNA polymerization. Thus, the most accurate DNA polymerase (pol-gamma) also incorporates the most nucleotides per association with the primer-template, while the least accurate enzyme (pol-beta) is the least processive. This correlation exists both for base substitution mutations and for single base frameshifts, and is most obvious for minus-one-base frameshifts in runs of pyrimidines. In support of this correlation, increasing the processivity of pol-beta from 1 to 4-6 incorporations per association increases the accuracy of in vitro DNA synthesis by severalfold. The data imply that the processivity of DNA synthesis could be an important factor in controlling the levels of spontaneous and perhaps induced mutations.     

High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12.

Slipped-strand mispairing (SSM) may play an major role in repetitive DNA sequence evolution by generating large numbers of short frameshift mutations within simple tandem repeats. Here we examine the frequency and size spectrum of frameshifts generated within poly-CA/TG sequences inserted into bacteriophage M13 in Escherichia coli hosts. The frequency of detectable frameshifts within a 40 bp tract of poly-CA/TG is greater than one percent and increases more than linearly with length, being lower by a factor of four in a 22 bp target sequence. The frequency increases more than 13-fold in mutL and mutS host cells, suggesting that a high proportion of frameshift events are normally repaired by methyl-directed mismatch repair. Of the 87 sequenced frameshifts in this study, 96% result from deletion or insertion of only or two 2 bp repeat units. The most frequent events are 2 bp deletions, 2 bp insertions, and 4 bp deletions, the relative frequencies of these events being about 18:6:1.     

Frameshift mutagenesis: the roles of primer-template misalignment and the nonhomologous end-joining pathway in Saccharomyces cerevisiae

Small insertions or deletions that alter the reading frame of a gene typically occur in simple repeats such as mononucleotide runs and are thought to reflect spontaneous primer-template misalignment during DNA replication. The resulting extrahelical repeat is efficiently recognized by the mismatch repair machinery, which specifically replaces the newly replicated strand to restore the original sequence. Frameshift mutagenesis is most easily studied using reversion assays, and previous studies in Saccharomyces cerevisiae suggested that the length threshold for polymerase slippage in mononucleotide runs is 4N. Because the probability of slippage is strongly correlated with run length, however, it was not clear whether shorter runs were unable to support slippage or whether the resulting frameshifts were obscured by the presence of longer runs. To address this issue, we removed all mononucleotide runs >3N from the yeast lys2ΔBgl and lys2ΔA746 frameshift reversion assays, which detect net 1-bp deletions and insertions, respectively. Analyses demonstrate that 2N and 3N runs can support primer-template misalignment, but there is striking run-specific variation in the frequency of slippage, in the accumulation of +1 vs. -1 frameshifts and in the apparent efficiency of mismatch repair. We suggest that some of this variation reflects the role of flanking sequence in initiating primer-template misalignment and that some reflects replication-independent frameshifts generated by the nonhomologous end-joining pathway. Finally, we demonstrate that nonhomologous end joining is uniquely required for the de novo creation of tandem duplications from noniterated sequence.     

Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment

Escherichia coli DNA polymerase IV (pol IV), a member of the error-prone Y family, predominantly generates -1 frameshifts when copying DNA in vitro. T-->G transversions and T-->C transitions are the most frequent base substitutions observed. The in vitro data agree with mutational spectra obtained when pol IV is overexpressed in vivo. Single base deletion and base substitution rates measured in the lacZalpha gene in vitro are, on average, 2 x 10(-4) and 5 x 10(-5), respectively. The range of misincorporation and mismatch extension efficiencies determined kinetically are 10(-3) to 10(-5). The presence of beta sliding clamp and gamma-complex clamp loading proteins strongly enhance pol IV processivity but have no discernible influence on fidelity. By analyzing changes in fluorescence of a 2-aminopurine template base undergoing replication in real time, we show that a "dNTP-stabilized" misalignment mechanism is responsible for making -1 frameshift mutations on undamaged DNA. In this mechanism, a dNTP substrate is paired "correctly" opposite a downstream template base, on a "looped out" template strand instead of mispairing opposite a next available template base. By using the same mechanism, pol IV "skips" past an abasic template lesion to generate a -1 frameshift. A crystal structure depicting dNTP-stabilized misalignment was reported recently for Sulfolubus solfataricus Dpo4, a Y family homolog of Escherichia coli pol IV.     

DNA replication errors produced by the replicative apparatus of Escherichia coli.

It has been hard to detect forward mutations generated during DNA synthesis in vitro by replicative DNA polymerases, because of their extremely high fidelity and a high background level of pre-existing mutations in the single-stranded template DNA used. Using the oriC plasmid DNA replication in vitro system and the rpsL forward mutation assay, we examined the fidelity of DNA replication catalyzed by the replicative apparatus of Escherichia coli. Upon DNA synthesis by the fully reconstituted system, the frequency of rpsL-mutations in the product DNA was increased to 1.9x10(-4), 50-fold higher than the background level of the template DNA. Among the mutations generated in vitro, single-base frameshifts predominated and occurred with a pattern similar to those induced in mismatch-repair deficient E. coli cells, indicating that the major replication error was slippage at runs of the same nucleotide. Large deletions and other structural alterations of DNA appeared to be induced also during the action of the replicative apparatus.     

Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila.

Deletions of specific DNA sequences are known to occur in Tetrahymena thermophila as a developmentally regulated process. Deletions of a particular region (region M) were previously shown to be of two alternative sizes, 0.6 or 0.9 kilobases (kb) (C.F. Austerberry, C.D. Allis, and M.-C. Yao, Proc. Natl. Acad. Sci. USA 81: 7383-7387). In this study, the nucleotide sequences for both deletions were determined. These two deletions share the same right junction, but their left junctions are 0.3 kb apart. An 8-base-pair (bp) sequence is present at both junctions of the 0.6-kb deletion, but only 5 bp of this direct repeat are present at the left junction of the 0.9-kb deletion. Further comparison revealed a common 10-bp sequence near each of the two left junctions and a similar sequence in inverted orientation near the right junction. These sequences may play a role in the developmental regulation of the deletion process.     

Chloroplast DNA replication in vitro: site-specific initiation from preferred templates.

An enzyme system prepared from maize chloroplasts catalyzes the synthesis of DNA from maize chloroplast DNA sequences cloned in bacterial plasmids. Cloned maize chloroplast DNA fragments Bam HI 17' (2470 bp) and Eco RI x (1368 bp) have been shown to be preferred templates for in vitro DNA synthesis catalyzed by pea chloroplast DNA polymerase preparations [Gold et al. (1987) Proc. Natl. Acad. Sci. USA 84, 194-198]. Analysis of replicative intermediates indicates that although the template activity of the recombinant plasmid pZmcBam 17' is substantially greater than that of the pZmcEco x, replication in both cases originates from within a 455 bp region which overlaps the two plasmids. The remaining approximately 1500 basepair portion of maize chloroplast BamHI fragment 17' is not more active because it contains additional origins for replication. The overlapping region shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that contains a replication origin. Replication is shown to proceed bidirectionally within the 455 bp origin region. Recombinant plasmid pZmc 427, which is also active in the in vitro DNA synthesis assay, promoted localized replication initiation within a 1 kbp Bg1II-Eco RI fragment of the chloroplast DNA insert, a region that includes the 3' terminal part of the psbA gene.     

The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3'' termini.

Our previous DNA sequence comparisons of 3' terminal portions from equivalent herpes simplex virus type 1 (HSV-1) and HSV-2 genes identified a conserved sequence (consensus YGTGTTYY; Y = pyrimidine) located approximately 30bp downstream from the AATAAA signal. We report here that this signal is located downstream from 67% of the mammalian mRNA 3' termini examined. Using constructions with the bacterial chloramphenicol acetyl transferase (CAT) gene linked to an HSV 'terminator' fragment, we show that deletions in the 'terminator' reduce CAT activities and the levels of CAT mRNA 3' termini. Specifically: (1) deletions of downstream sequences which extend up to the consensus YGTGTTYY signal reduce CAT levels to values 35% of those obtained with undeleted plasmids, (2) a deletion of a further 14bp, which removes the YGTGTTYY consensus but not the poly A site, reduces CAT activities to 1%-4%. The levels of CAT mRNA 3' termini reflect the reductions in CAT activities however, levels of mRNA 5' termini are unaffected by these deletions. The RNA produced in the absence of the YGTGTTYY signal is present in the cytoplasm although no CAT activity is detectable.     

Bridging PNAs can bind preferentially to a deleted mitochondrial DNA template but replication by mitochondrial DNA polymerase gamma in vitro is not impaired

Mutations in mitochondrial DNA (mtDNA) are an important cause of neurological and other human pathologies. In the vast majority of cases, supportive care only is available. Mutated and wild-type mtDNAs often coexist in the same cell. A strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Peptide nucleic acid molecules (PNAs) can be designed to selectively target pathogenic mtDNA with single point mutations. Molecules harbouring deletions present a complex problem. Deletions often occur between two short repeat sequences (4-13 residues), one of which is retained in the deleted molecule. With the more common large repeats, it is therefore difficult to design an antigenomic molecule that will bind selectively under physiological conditions. Following limited success with antigenomic oligodeoxynucleotides (ODNs), we have repeated these studies with a series of bridging PNAs. Molecules complementary to the sequence flanking either side of the 13 bp 'common deletion' were synthesised. The PNAs demonstrated markedly greater affinity for the delete than to the wild-type template. In runoff assays using Klenow fragment, these PNAs selectively inhibited replication of the delete template. However, no selective inhibition was observed when a polymerase gamma-containing mitochondrial fraction was used.     

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.