In both human and mouse, 25% of TUs resided in cis–antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis–antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis–antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248  cis–antisense pairs) or start from a genome region  at which transcription can initiate in both directions along the DNA. In this paper, researchers present the one of the most comprehensive censuses of complex loci to date and investigate their general properties and human–mouse differences to discover the rules of this type of gene organization and its effect on gene regulation. They found about 25% of known human and mouse genes to be in cis–antisense pairs, and estimate the total fraction to be over 40%. At bidirectional promoters, they demonstrated the existence of mirror DNA sequence composition related to the promoters' ability to initiate transcription in two directions. The researchers found over 2,000 “chains”—complex arrangements where three or more genes are coupled by cis–antisense pairing and/or bidirectional promoters; among them are many genes whose products control the expression of other genes.In the traditional view, most genes occupy their own distinct territory in mammalian genomes. However, it has become apparent that many genes are in fact located in complex regions (complex loci) where they share territory with other genes by utilizing opposite strands of DNA. Such genes either share regions expressed as mRNA (i.e., form  Several recent reports indicate that the transcriptional complexity of mammalian genomes has been significantly underestimated. Large-scale sequencing of full-length transcripts, expressed sequence tags (ESTs), and shorter tags  and trancis-coupling of genes have been reported to be widespread in mammalian genomes. (1) More than a thousand pairs of divergently transcribed, nonoverlapping genes spaced by less than 1,000 bp have been found in the human genome, comprising 9% of known genes [cis–antisense pairs) have been identified in human and mouse genomes [cis–antisense pairs [cis–antisense pairs might be even more widespread, perhaps involving more than 60% of all loci [cis–antisense pairs, there is evidence that paired genes tend to be coexpressed [cis–antisense pairs can regulate expression of the gene encoded on the opposite strand . Numeroiew, see ,18).cis–antisense pairs where the genes have orthologs in pufferfish, proximity and orientation of the paired genes is conserved in pufferfish significantly more often than for pairs of neighboring genes on the same strand [cis–antisense pairs in both organisms have previously reported at most 347 gene pairs [cis–antisense pairs have been reported to lack elevated conservation in general [cis–antisense pairs might have been their exclusive focus on protein-coding genes: recent unbiased surveys of mouse transcripts have indicated that cis–antisense pairs most frequently consist of one coding and one noncoding transcript [Cross-species genome comparisons can reveal conserved genomic features that are likely to be functionally important, and species-specific features that might underlie phenotypic differences. For the great majority (81%) of human bidirectionally promoted pairs where the genes have mouse orthologs, the bidirectional arrangement is conserved, suggesting that it is functionally important . Similare strand . Howeverne pairs –21, a ve general , contrar general . A limitanscript ,13.cis–antisense and bidirectionally promoted gene pairs from human and mouse transcript sequence data and present an analysis of the higher-level organization of these pairs in complex loci. We further explore human–mouse conservation of complex loci at both sequence and structure levels, taking into account both coding and noncoding transcripts. We describe a widespread occurrence of “chains” of overlapping transcriptional units (TUs), a several times greater number of human–mouse conserved cis–antisense pairs than previously reported, and additional species-specific complex arrangements. We perform sampling to reach an estimate of the total fraction of genes in cis–antisense arrangement, and of the fraction of such arrangements that are conserved between human and mouse. We study the sequence composition of bidirectional promoters and its relation to the positioning of transcriptional start sites (TSSs). Finally, we take a closer look at a number of homeotic genes, to assess the extent of transcription from the opposite strand at these loci and the conservation of the transcripts.We define complex loci as genomic regions in which multiple genes share transcribed regions in antisense orientation and/or bidirectional core promoters. In this study we construct comprehensive and highly reliable genome-wide datasets of We inferred TUs from genomic mappings of EST and full-length cDNA sequences from FANTOM3 and the public databases. Particularly rigorous criteria were applied to thoroughly eliminate the inclusion of artificially reversed sequences see . This isStarting from 161,805 human and 140,769 mouse cDNA sequences and a total of ~8 million ESTs, we obtained 42,887 human and 36,606 mouse TUs . Using tcis–antisense pairs, non-exon-overlapping antisense pairs, and bidirectionally promoted pairs  were supported by sequence types generally considered to be of high quality (either cDNA or spliced EST mappings), indicating that our set of cis–antisense pairs is highly reliable.To further investigate the reliability of the overlaps . Most pacis–antisense pair dataset, we performed orientation-specific RT-PCR as previously described [cis–antisense pairs supported by at least one cDNA or EST from adult mouse brain on each strand. As negative controls, we selected five highly expressed genes for which we could find no evidence of antisense transcription in sequence databases. We were able to detect the coexpression of sense and antisense transcripts in brain for 16 of the 20 cis–antisense pairs ((Rps27) also reproducibly showed evidence of antisense transcription. In retrospect, this control was ill-chosen: there are several copies of Rps27 pseudogenes in the mouse genome, so the complementary transcripts need not be transcribed from the same loci as Rps27 itself. In conclusion, the RT-PCR results suggest that at least 80% of the cis–antisense pairs in our dataset are expressed from both strands, and that there might exist a significant number of antisense transcripts that are yet to be discovered.To experimentally assess the validity of our escribed ,24. We ise pairs . For onecis–antisense pairs in our set (34% of human pairs and 34% of mouse pairs) had multiple distinct exon-to-exon overlaps. Both genomes had on average 1.6 distinct exon-to-exon overlaps per cis–antisense pair. The size distributions for exon overlaps ranged from 1 bp to 5,200 bp with medians of 159 bp (human) and 172 bp (mouse), and distinct peaks around 100 bp. The average repeat content within exon overlaps was 9.5% in human and 5.9% in mouse. For comparison, we measured the repeat content within the entire exonic sequence of each TU. The average repeat content of entire TUs was nearly three times as high for TUs in general , and nearly two times as high for TUs involved in cis–antisense pairs . We therefore concluded that cis–antisense pairs tend to involve TUs with low repeat content, and that exon overlaps tend to be located in repeat-poor regions of those TUs. However, for a subset of cis–antisense pairs , more than 90% of the exon overlap was repeat sequence. (Here it should be noted that there might be an underrepresentation of repeat-rich transcripts in the dataset because of the inherent difficulty of unambiguously mapping them onto the genome.)Many cis–antisense pairs—based on transcriptional direction of participant TUs—as divergently transcribed (head-to-head overlapping), convergently transcribed (tail-to-tail overlapping), or fully overlapping (one TU completely spanned by the other). In agreement with previous observations on the FANTOM3 dataset [cis–antisense pairs were also roughly equally common, but fully overlapping pairs were more frequent, constituting 42% of all pairs. A significant number of these fully overlapping pairs might represent actual divergent/convergent cases that were not detected as such because of the lower availability of full-length cDNA sequence for human.We classified the  dataset , we founcis–antisense pairs, we recomputed the TU and cis–antisense pair datasets using random subsets of all available transcript sequences. cis–antisense pairs as a function of the number of transcript sequences used. For both human and mouse, a saturation curvea is the fraction of TUs involved in cis–antisense pairs at saturation, and c  is a measure of sequence redundancy in the set and depends on the choice of sampled set . The saturation curves predicted that the fraction of TUs involved in cis–antisense pairs approaches 0.45 for human and 0.43 for mouse as the number of transcript sequences increases. Using two other sampling approaches, we obtained closely similar estimates  Genome Browser Database [cis–antisense pairs, respectively (cis–antisense pairs (69% of human pairs and 82% of mouse pairs) had more than 90% of their exon overlap sequence within BLASTZ net alignments, indicating that the implicit requirement for sequence similarity imposed by the use of precomputed alignments did not severely limit our ability to detect conserved cis–antisense pairs. However, it is likely that a large number of truly conserved pairs were not detected as such because of transcript sequences that have not been discovered yet. We attempted to estimate the true extent of conservation of cis–antisense pairs by a sampling approach equivalent to the one we employed above to estimate the fraction of TUs involved in cis–antisense pairs. To estimate how observed conservation grows with increasing transcript sequence data, we compared the entire human dataset against mouse datasets computed from different-sized random samples of all available mouse transcript sequences  was preferable as it provided an equally good fit while being simpler. The curve predicts that up to about 25% of human cis–antisense pairs are conserved in mouse per the definition of conservation employed here. To estimate whether mouse cis–antisense pairs are likely to be conserved at a similar rate in human, we repeated the analysis in an analogous manner, sampling human transcripts instead of mouse transcripts. A hyperbolic saturation model again fit well to the data and predicted that about 26% of mouse cis–antisense pairs are conserved in human at the saturation level (unpublished data).As noted above, we found a striking agreement between human and mouse in prevalence and general properties of Database ; see Matectively . The humequences C. The sacis–antisense pairs are not conserved between human and mouse. Accordingly, detailed inspection of homeotic and other transcription factor loci provided several examples of nonconserved cis–antisense and bidirectionally promoted transcripts, some with experimentally supported regulatory roles (see below). We therefore wanted to examine the genome-wide occurrence of nonconserved transcripts in cis–antisense or bidirectionally promoted arrangement with known genes. To find such TUs in the cis–antisense pair dataset, we focused on the subset of cis–antisense pairs where one member (the known gene) had detectable conservation outside the region of antisense overlap, and the other member (the nonconserved TU) showed no conservation outside the region of overlap. Among all 3,442 divergent and convergent cis–antisense pairs in mouse  Thus, we identified a total of 90 nonconserved TUs in bidirectionally promoted or cis–antisense arrangement with known genes.Our saturation estimates indicated that most in mouse , there wcis–antisense pairs, and a further 17 sharing bidirectional promoters with known genes . In addition, CpG islands associated with bidirectional promoters were significantly larger than CpG islands associated with unidirectional promoters . To experimentally confirm the observed size of transcriptional initiation regions in bidirectional promoters, we used quantitative real-time PCR (qRT-PCR) to measure expression levels in mouse brain RNA samples of different regions near the 5′-ends of transcripts from the genes Ddx49 and Cope, which share a bidirectional promoter  data has confirmed two major types of TSS regions associated with different types of core promoters . TATA-box promoters typically initiate transcription from a single position in the genome, while TATA-less promoters can initiate transcription within an interval of 100 bp or more that often coincides with a CpG island . We assepromoter . For DdxThe two divergently oriented transcription start regions identifying a bidirectional promoter were generally closely spaced, but for only 12% of bidirectional promoters did the TCs overlap by one or more bases . To inveCpG islands often contain multiple binding sites for the transcription factor Sp1 . Considecis–antisense pairs with regulatory interactions between pair members, we concentrated on 242 pairs from mouse with available microarray expression data for 61 tissues  of pairs with available probesets were convergently (tail-to-tail) overlapping. We found a significant positive correlation across the entire panel of tissues for 58/242 (24%) cis–antisense pairs at the 0.05 level, and a significant negative correlation for only 14/242 (6%) pairs. After correcting for multiple testing, 17/242 (7%) pairs remained significantly positively correlated, and no pairs remained significantly negatively correlated. We assessed how likely it would be to obtain this result if TUs were paired at random. In only three out of 10,000 sets of 242 random TU pairs did we obtain 17 or more significantly correlated pairs, and none of the sets contained more than 14 significant positive correlations . We selelations . By the nt class . For 15 nt class , indicat overlap . The lare repair .cis–antisense pairs will always have an average density higher than that of individual random genes, we examined 100-kbp regions directly flanking each pair, rather than the region covered by the pair itself. Regions flanking cis–antisense pairs had roughly 30% higher TU density and 30% more exon sequence than regions flanking TUs not involved in cis–antisense pairs  and three metabolic genes, allowing the possibility of cis-regulation of genomically adjacent genes of diverse functions and resulting effects on their downstream targets. The genomic distribution of chains, cis–antisense pairs, and bidirectionally promoted pairs is illustrated in cis–antisense pairs, bidirectionally promoted pairs, and chains.Next we investigated whether local gene density was related to the incidence of antisense transcription. To avoid bias due to the fact that se pairs . Many TUHOXA11 gene have been found to be conserved between human and mouse [HOXA cluster in both organisms [Hoxa3 and Hoxa7 genes formed a chain together with two uncharacterized TUs. In human, there were three chains of three TUs each, including HOXA3, HOXA4, HOXA9, HOXA10, HOXA11, and four uncharacterized TUs. We have further shown that several noncoding transcripts in the human HOXA cluster are coexpressed with adjacent coding HOXA genes in various human tissues, and are likely to be involved in the opening and closing of chromatin and sequential transcriptional activation of HOX cluster members . Based on these findings, we selected homeotic genes as a group to focus on. HOX genes are arranged into four clusters in both human and mouse genomes [HOX genes are transcribed in the same direction. To assess the extent of transcription from the opposite strand at HOX loci and around dispersed homeotic genes , we specifically searched for EST sequences that mapped to the opposite strand at such loci and either overlapped homeotic genes or were intergenically located. We detected 232 human and 46 mouse ESTs on the opposite strand at HOX loci  displayed ESTs with a high degree of sequence similarity. These matches did not span entire transcripts, but in each case were limited to a portion of a single exon. Given that HOX gene clusters and other homeotic gene loci are known to be spanned by arrays of highly conserved putative regulatory elements [ESTs at corresponding locations in human and mouse  (HOXA11 ) was a selements ,34, thesAdditionally, we manually inspected conservation in human of 234 mouse chains containing genes for other transcriptional regulatory proteins. Consistent with the homeotic genes case study, the results suggest lack of association between sequence conservation of chained transcriptional regulators and structure conservation of their chains see .cis–antisense pairs in the human genome and 5,248 in the mouse. While methodological differences in redundancy reduction and clustering preclude direct comparison to earlier estimates also based on cDNA and EST data [cis–antisense pair counts compared to these previous reports and widespread chaining of bidirectional transcription indicate that the earlier studies underestimated the prevalence and complexity of antisense transcription in human.As part of our effort to characterize complex loci, we report to our knowledge the most comprehensive list to date: 6,141 EST data ,12,21, tTaking advantage of the newly available CAGE tag data on TSSs , we havecis–antisense transcription with up to 11 TUs per chain. We identified 13 chains whose structures were entirely conserved between human and mouse, and that contained genes encoding transcriptional regulators  have trans-regulatory impact when chains contain transcription factor genes. The structure of the complex loci also indicates that they should be taken into account in the process of designing microarray probes, which for the purpose of assessing expression levels of individual TUs in these loci should both be strand-specific and avoid targeting sequences shared by multiple TUs.There is a paucity of previous studies on genes organized into structures that we refer to as chains. Veeramachaneni et al.  presentegulators : these ccis–antisense and bidirectionally promoted pairs, as well as the structural properties of cis–antisense overlaps. The amount of sequence evidence that supports the existence of these structures is sufficient to reject the explanation that these transcripts are due to methodological “noise” [cis–antisense pairs are conserved between human and mouse is in agreement with earlier data [cis–antisense pairs we report here are close to seven [cis–antisense pairs, which is about 40% both in human and in mouse, regardless of the sampling method, sequence dataset redundancy, and different average quality of EST and full-length cDNA sequence data between the two species. Even though there exists a possibility that an even higher number of unsampled noncoding transcripts are present, the clear saturation of the sampling plot, the number of ESTs, and the diversity of sampled libraries make it unlikely that our method seriously underestimates the total count. Our estimate is in agreement with a recent report where hundreds of novel transcripts were characterized by RACE, and where it was found that 44% of all investigated transcripts overlapped a transcript on the opposite strand [There is a striking agreement between the human and mouse datasets regarding proportions of TUs involved in  “noise” . Our finier data –21,24. Hto seven  or threeHOX genes are master regulators of vertebrate development and differentiation. Even though the loci of the four HOX gene clusters contain abundant evidence of transcription from the opposite strand in both human and mouse, we were unable to detect significantly evolutionary conserved antisense ESTs. This is unlikely to be entirely due to incomplete EST coverage of the regions, given the depth of recent EST sequencing efforts [HOXA locus are induced upon retinoic acid treatment, following the timely colinearity of the HOX sense transcripts . The results suggest that antisense-strand transcription is involved in the opening and activation of mammalian HOX clusters and prevents, as an anti-silencing mechanism, the re-repression of the cluster. Therefore, transcription at a fixed location but without a fixed sequence can be functionally relevant to regulation of ancient conserved gene clusters fundamental to vertebrate development, implying that nonhomeotic positional equivalents, similarly, may impact the expression of their paired conserved adjacent genes. efforts ,35 and tcis–antisense and bidirectionally promoted arrangement show a higher probability of being coordinately expressed than random pairs of genes, our overall understanding of their coregulation remains lacking. Current publicly available microarray datasets are of limited utility for studying the expression of overlapping genes, because of uncertainties about whether probes outside of cis–antisense overlaps sufficiently well represent transcript isoforms comprising the cis–antisense pairs, the possibility of mixed-signal detection unless expression measurements are strand-specific, and the limited representation of noncoding genes on commercially available microarrays.Even though we managed to reproduce the previous findings ,15 that In conclusion, we have shown that complex loci are widespread and include numerous lineage-specific transcripts and nonconserved gene structures. They are likely involved in regulatory events affecting large numbers of transcription factor genes, and are also associated with locus-specific synergistic expression profiles of paired genes. While many questions about the complex loci remain open, our complex loci catalog establishes a foundation for querying the regulatory significance of complex loci components by strand-specific microarray-based expression analyses ,37, targt. The threshold t was set to the smallest integer greater than two for which P ≥ t) < 0.01 . The rationale behind the above threshold calculation was that, to infer a TU from unspliced ESTs only, we wanted the probability that those ESTs are misoriented (and therefore should belong to other-strand TUs) to be less than 0.01. The thresholding eliminated 128 potential TUs supported by more than three mappings.Mappings of all public human cDNA and EST sequences to human genome assembly hg17 were obtained from the UCSC Genome Browser Database  in OctobWe used spliced mappings that could be unambiguously oriented by their splice signals to estimate the accuracy of the part of the orientation procedure that handles unspliced mappings . All maphttp://www.invitrogen.com) in accordance with the manufacturer's protocol. qRT-PCR was carried out with first-strand cDNA corresponding to 12.5 ng of total RNA per test well using the tailor-made reaction [http://www.appliedbiosystems.com) using the following cycling protocols: 15-min hot start at 94 °C, followed by 40 cycles of 15 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C. The threshold cycle (Ct) value was calculated from amplification plots, in which the fluorescence signal detected was plotted against the PCR cycle. The number of transcripts was calculated from the slope of the standard curve using genomic DNA or the corresponding cDNAs. Averages and standard deviations were calculated based on six qRT-PCR measurements for each primer pair.Adult male C57BL/6J mice were killed according to the RIKEN Institute's guidelines and the tissues were removed. Total RNA was extracted by the acid phenol-guanidinium thiocyanate-chloroform method . RNA wasreaction . The PCRcis–antisense pair to be conserved in mouse if it had a region of exon overlap that aligned with a region of exon overlap from a mouse cis–antisense pair over at least 20 bp in both human-to-mouse and mouse-to-human BLASTZ net alignments. Saturation curves were fitted using nonlinear regression (nls function) in R (http://www.R-project.org).For the comparative analyses, alignments between the human and mouse genomes  were retrieved from the UCSC Genome Browser Database . Human-tcis–antisense exon overlap. Regions where exons overlapped were excluded from consideration, since conservation in such regions cannot be specifically attributed to one of the pair members. We required that one pair member have exon sequence that overlapped with a BLASTZ tight alignment, and the other pair member have no exon sequence that overlapped with a BLASTZ net alignment. We further eliminated pairs where the “nonconserved” member gave a significant BL2SEQ alignment  [Candidate pairs for manual curation were selected by assessing the conservation of exon sequence outside regions of ter off)  with theTCs were defined by associating CAGE tag mappings that overlapped on the genome  . To study the locations of TSSs at bidirectional promoters, we constructed a set of 766 bidirectional promoters defined by TCs by scanning the mouse genome for pairs of divergently oriented TCs spaced by less than 500 bp and having no intervening genome assembly gaps. Divergent TCs were allowed to overlap only partially, i.e., the most downstream TSS in each TC was required to be outside the overlap. We only considered TCs that contained at least ten tags and overlapped the 5′-end of a TU or cDNA sequence approved by the TU inference pipeline. To avoid redundancy, we paired a TC only with the nearest divergent TC satisfying these criteria. A control set of unidirectional promoters was constructed by scanning for single TCs fulfilling the same criteria for tag counts and TU/cDNA overlap and that had no assembly gaps or divergently oriented TCs with any number of tags in the 500 bp upstream.Using locations of CpG islands obtained from the UCSC Genome Browser Database , a TC wahttp://wombat.gnf.org). cis–antisense pairs in mouse were matched with GNF1M probesets using probe-to-FANTOM cDNA clone mappings produced in the FANTOM3 collaboration [cis–antisense pairs we selected only probesets that mapped to regions of exon overlap between the cis–antisense partners. When multiple probesets were available for a TU, the probeset mapping to the most cDNA sequences was selected in order to preferentially measure major transcript isoforms. Spearman rank correlations and p-values were computed for each pair over all tissues, using the function cor.test in R. p-Values were adjusted for multiple testing using the Bonferroni method.MAS5-processed expression data for 61 mouse tissues measured in duplicate on the GNF1M chip  were obtcis–antisense overlaps and/or putative bidirectional promoters. In order not to overestimate the occurrence and extent of chains, we did not allow chains to be held together by (1) gaps between EST end-reads, (2) other gaps that separated exons and were not classified as introns by the criteria described above, (3) introns with GC/AG or AT/AG splice signals and longer than 15 kb, and (4) introns with GT/AG splice signals and longer than 45 kb. Cases 2 to 4 were allowed if supported by more than one mapping. When counting the number of TUs in a chain, we ignored unspliced TUs that started within 1.5 kb of the end of a mapping that were on the same strand and part of the same chain, since such unspliced TUs might represent 3′-UTR extensions. We note that this rule might be overly conservative in light of the recent discovery of widespread transcription initiation within 3′-UTRs [Chains were identified by searching the entire TU dataset for clusters of three or more TUs connected by ether by  gaps betHOX clusters and 10-kb regions centered on TSSs of dispersed homeotic genes for presence of antisense-strand transcripts using the AntiHunter software [E-value below 0.001 were considered significant. Genomic comparisons between human and mouse loci were performed using PipMaker [The precise genomic organization of homeotic loci was determined by visualizing them in the UCSC Genome Browser  and by Bsoftware  with defsoftware . Human asoftware  with defPipMaker  with defcis–antisense pairs, bidirectionally promoted pairs, and chains can be obtained at http://www.genereg.net/complex_loci. A detailed image is provided for each chain.The dataset of TUs, Figure S1(32 KB PDF)Click here for additional data file.Figure S2(191 KB PDF)Click here for additional data file.Figure S3(30 KB PDF)Click here for additional data file.Figure S4(319 KB PDF)Click here for additional data file.Figure S5(66 KB PDF)Click here for additional data file.Figure S6(14 KB PDF)Click here for additional data file.Figure S7(6.4 MB TIF)Click here for additional data file.Table S1(28 KB PDF)Click here for additional data file.Table S2(590 KB PDF)Click here for additional data file.Table S3(25 KB PDF)Click here for additional data file.Table S4(25 KB PDF)Click here for additional data file.Table S5(17 KB PDF)Click here for additional data file.Table S6(18 KB PDF)Click here for additional data file.Table S7(19 KB PDF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) accession numbers for the genes discussed in this paper are Bop1 (12181), Cope (59042), D330001F17Rik (223658); Ddx49 (234374), Dgat1 (13350), DLX6 (1750), HOXA10 (human) (3206), HOXA11 (human) (3207), HOXA3 (human) (3200), Hoxa3 (mouse) (15400), HOXA4 (human) (3201), Hoxa7 (mouse) (15404), HOXA9 (human) (3205), Hsf1 (15499), OTX2 (5015), PITX1 (5307), Ppp1ca (19045), Rad9 (19367), RNH1 (human) (6050), Rnh1 (mouse) (107702), Rps2 (16898), Rps27 (57294), TCF8 (6935), and titin (7273).The NCBI EntrezGene (


Research conducted in recent years has increased public health concern about the toxicity of lead at low dose and has supported a reappraisal of the levels of lead exposure that may be safely tolerated in the workplace. In this article, which appears as part of a mini-monograph on adult lead exposure, we summarize a body of published literature that establishes the potential for hypertension, effects on renal function, cognitive dysfunction, and adverse female reproductive outcome in adults with whole-blood lead concentrations < 40 μg/dL. Based on this literature, and our collective experience in evaluating lead-exposed adults, we recommend that individuals be removed from occupational lead exposure if a single blood lead concentration exceeds 30 μg/dL or if two successive blood lead concentrations measured over a 4-week interval are ≥ 20 μg/dL. Removal of individuals from lead exposure should be considered to avoid long-term risk to health if exposure control measures over an extended period do not decrease blood lead concentrations to < 10 μg/dL or if selected medical conditions exist that would increase the risk of continued exposure. Recommended medical surveillance for all lead-exposed workers should include quarterly blood lead measurements for individuals with blood lead concentrations between 10 and 19 μg/dL, and semiannual blood lead measurements when sustained blood lead concentrations are < 10 μg/dL. It is advisable for pregnant women to avoid occupational or avocational lead exposure that would result in blood lead concentrations > 5 μg/dL. Chelation may have an adjunctive role in the medical management of highly exposed adults with symptomatic lead intoxication but is not recommended for asymptomatic individuals with low blood lead concentrations. In deriving the recommendations in this article, we took note of a body of literature that establishes the potential for adverse health effects at blood lead concentrations or exposure levels permissible under current workplace regulations established in the 1970s by the U.S. Occupational Safety and Health Administration (OSHA). These regulations generally require removal from lead exposure when whole-blood lead concentrations exceed 50 or 60 μg/dL. These values are considerably above blood lead concentrations of the general population of the United States, which had a geometric mean of 12.8 μg/dL in the late 1970s , the average blood lead concentration was 6.3 μg/dL. On the basis of the subjects’ ages (mean 67 ± 7.2 years), it may be expected that they lived most of their adult lives at a time when the blood lead concentration of the general population ranged from 10 to 25 μg/dL , the subgroup with blood lead concentration ≥ 10 μg/dL  had a relative risk of cardiovascular mortality of 1.59  compared with subjects with blood lead < 5 μg/dL , the relative risk for cardiovascular mortality was 1.53 , comparing a blood lead of 4.92 μg/dL (80th percentile of the distribution) with a blood lead of 1.46 μg/dL (20th percentile of the distribution)  .N-acetyl-β-d-glucosaminidase (NAG), a biomarker of early biological effect on the renal tubule, but in an analysis of a smaller subset of the lead workers (n = 190) that controlled for the relatively low levels of urinary cadmium (1.1 ± 0.78 μg/g creatinine), only the relationship with tibia lead and NAG remained significant , and between cumulative blood lead index and NAG   .n = 744), there was a negative correlation between blood lead  and measured creatinine clearance, after natural log transformation of both variables and adjustment for other covariates ; n = 1,016 women ], log-transformed blood lead concentration was inversely correlated with measured creatinine clearance (n = 820), low levels of blood lead  were inversely correlated with creatinine clearance and glomerular filtration rate, after adjusting for age, body mass index, urinary or blood cadmium, hypertension, diabetes, and regular use of nonsteroidal anti-inflammatory drug (NSAID) medication . In a cross-sectional investigation of a subcohort of middle-aged to elderly men enrolled in the Normative Aging Study , blood lead was a risk factor for elevated serum creatinine  and “chronic kidney disease” (defined as an estimated glomerular filtration rate < 60 mL/min) only among subjects with hypertension (n = 4813) , multiple regression analysis revealed that log-transformed blood lead was positively correlated with serum creatinine in hypertensive but not normotensive subjects  . Comparesubjects . In a losubjects . An intesubjects . Althougn ≤ 100) with blood lead concentrations ranging approximately 20–40 μg/dL have associated lead exposure with subclinical decrements in selective domains of neurocognitive function (n = 803 workers) (n = 576 workers) (n = 48) and age-matched controls with similar blood lead concentrations , increases in current blood lead concentration within the entire study population were correlated with poorer performance on several tests of neurocognitive function but on only one measure was cumulative lead exposure (measured in the workers) associated with poorer performance   and a 3-workers)  found thformance .n = 4937), there was no relationship between blood lead concentration  and covariate-adjusted performance on neurocogntive function (n = 325) with background, community lead exposure , certain measures of neuropsychologic function (Trailmaking part B and Digit Symbol test) were performed more poorly by women in the upper 15th percentile of blood lead , no relationship between blood lead  and neuropsychologic performance was discernible  with a mean blood lead concentration of 5.5 ± 3.5 μg/dL examined as part of the Normative Aging Study, increased blood lead concentration was associated with poorer performance on neuropsychologic assessment of memory, verbal ability, and mental processing speed  from the Normative Aging Study assessed with the Mini-Mental Status Examination (MMSE), the OR for having a test score associated with an increased risk of dementia was 3.4  comparing the mean blood lead of the highest quartile  to that of the lowest quartile  (In the population-based sample of adults 20–59 years of age participating in the NHANES III study (function . Howevercernible . In a geng speed . In a la5 μg/dL) . There wn = 466; mean age, 67.4 ± 6.6 years) examined for longitudinal change in MMSE score over an average of 3.5 ± 1.1 years, higher patella bone lead concentrations, a biomarker of cumulative lead exposure, predicted a steeper decline in performance . Although several earlier studies failed to detect this substantial impact, they may have been subject to methodologic limitations not present in the Mexico City investigation , every increase of 10 μg/g in maternal tibia lead was associated with a 73-g  decrease in birth weight . The impPrenatal lead exposure assessed by umbilical cord blood lead concentration has been inconsistently associated with an adverse effect on neurobehavioral development in childhood. However, recent studies suggest that mobilization of maternal bone lead during pregnancy may contribute to fetal lead exposure in ways that may be incompletely reflected by the single measurement of umbilical cord whole-blood lead . In a prn = 119). Adjusting for covariates that included maternal age, maternal IQ, child sex, childhood weight and height for age, and childhood whole-blood lead at 24 months, an increase of one SD in loge –transformed plasma lead in the first trimester was associated with a 3.5-point decrease in score on the 24-month MDI of the Bayley Scales of Infant Development. The corresponding impact of one SD increase in loge maternal whole blood during the first trimester was a 2.4-point decrease in the 24-month MDI. The logarithmic relationship between maternal plasma and blood lead concentrations and infant MDI indicated that the strongest effects occurred among mothers with the lowest plasma and blood lead concentrations.A prospective study that measured maternal plasma lead and maternal whole-blood lead during pregnancy found that maternal plasma lead during the first trimester was the stronger predictor of infant mental development at 24 months of age . In thisn = 390) (n = 150); from 1 through 5 years it was 9.8 μg/dL (2.8–36.4 μg/dL), and from 6 through 10 years it was 6.2 μg/dL . IQ at 6 to 10 years of age, assessed by the Wechsler Intelligence Scale for Children—Revised, decreased significantly only with increasing natural-log third-trimester blood lead, controlling for other blood lead measurements and covariates. Every doubling of third trimester blood lead  was associated with an IQ decrement of 2.7 points . Notably, the nonlinear  relationships detected in the Yugoslavia and Mexico City studies indicate that across a maternal blood lead range of 1–30 μg/dL, an increase in blood lead from 1 to 10 μg/dL will account for more than half the IQ decrement.Two long-term prospective studies that conducted multiple measurements of maternal blood lead during pregnancy and childhood have identified an adverse impact of low-level prenatal lead exposure on postnatal neurobehavioral development extending beyond infancy. Applying a repeated measures linear regression technique to analysis of age-appropriate IQ test data obtained in 390 children 3–7 years of age, the Yugoslavia Prospective Lead Study found independent adverse effects of both prenatal and postnatal blood lead. After controlling for the pattern of change in postnatal blood lead and other covariates, IQ decreased 1.8 points  for every doubling of prenatal blood lead, which was assessed as the average of maternal blood lead at midpregnancy and delivery  . The Mexn = 390) . Geometrn = 637) without regard to immigration status found that every 10-μg/g increase in calcaneus (heel) bone lead increased the OR of third trimester pregnancy hypertension (systolic > 90 and/or diastolic > 140 mmHg) by 1.86   were associated with increased systolic blood pressure during labor (1.0 mmHg for every doubling of blood lead) and increased odds of hypertension (not further defined) recorded any time during pregnancy  for every doubling of blood lead . A prosp04–3.32) .3 as an 8-hr time-weighted average, the recommendations in The OSHA workplace standard for lead exposure in general industry (adopted in 1978) and a corresponding standard for lead exposure in construction trades (adopted in 1993) set forth medical surveillance requirements that include baseline and periodic medical examinations and laboratory testing. Details of the two standards, which establish distinct criteria for the implementation of surveillance, can be found on the OSHA website . BecauseThe content of the baseline or preplacement history and physical examination for lead-exposed workers should continue to follow the comprehensive scope set forth in the OSHA lead standard for general industry. Measurement of serum creatinine will identify individuals with chronic renal dysfunction who may be subject to increased health risks from lead exposure. With the potential exception of an annual blood pressure measurement and a brief questionnaire regarding the presence of medical conditions  that might increase the risk of adverse health effects of lead exposure, medical evaluations for lead-exposed workers should be unnecessary as long as blood lead concentrations are maintained < 20 μg/dL. Annual education of lead workers regarding the nature and control of lead hazards, and ongoing access to health counseling regarding lead-related health risks are recommended as preventive measures.As summarized earlier in this article, the recent findings concerning lead-related adverse reproductive outcomes render it advisable for pregnant women to avoid occupational or avocational lead exposure that would result in blood lead concentrations > 5 μg/dL. Calcium supplementation during pregnancy may be especially important for women with past exposure to lead. Calcium decreases bone resorption during pregnancy  and may Maternal body lead burden and external lead exposure influence the lead concentration of breast milk . The fewRemoval from all sources of hazardous lead exposure, whether occupational or nonoccupational, constitutes the first and most fundamental step in the treatment of an individual with an elevated blood lead concentration. A careful history that inquires about a broad spectrum of potential lead sources is recommended . RemovalMedical treatment of individuals with overt lead intoxication involves decontamination, supportive care, and judicious use of chelating agents. Comprehensive discussion of such treatment is beyond the scope of this article but has been reviewed in recent medical toxicology texts , 2005. AIn our experience, adults with blood lead concentrations ≥ 100 μg/dL almost always warrant chelation, as levels of this magnitude are often associated with significant symptoms and may be associated with an incipient risk of encephalopathy or seizures. Occasionally, patients with very high blood lead concentrations may have no overt symptoms. Patients with blood lead concentrations of 80–99 μg/dL, with or without symptoms, can be considered for chelation treatment, as may some symptomatic individuals with blood lead concentrations of 50–79 μg/dL. These demarcations are imprecise, however, and decisions on chelation should be made on a case-by-case basis after consultation with an experienced specialist in occupational medicine or medical toxicology.Hair lead analysis or measurement of urine lead concentration seldom provide exposure information of clinical value beyond that provided by the history and the measurement of blood lead concentration. Chelation initiated exclusively on the basis of hair or urine lead levels or chelation of asymptomatic individuals with low blood lead concentrations is not recommended.Adults with overt lead intoxication will generally experience improvement in symptoms after removal from lead exposure and decline in blood lead concentration. This clinical observation on improvement in overt symptoms finds some support from the relatively limited number of studies that have examined the impact of naturally declining blood lead concentrations on cognitive function in occupationally exposed subjects . ImproveWith appropriate engineering controls, safe work practices, and personal protective equipment, workers without a previous history of substantial lead exposure should be able to work with lead in a manner that minimizes the potential for hazardous levels of exposure. For such workers, elevations in blood lead concentration that result from unforeseen transient increases in exposure will often decline promptly once the exposure is controlled. However, in a worker with a long history of high exposure, redistribution of lead from a large internal skeletal burden may result in a prolonged elevation of blood lead concentration despite marked reductions in external lead dose.The recommendations for management of adult lead exposure contained in this article are derived from consideration of risks to health, and have not been the subject of a cost-benefit analysis examining economic feasibility or social impacts. Nonmedical, socioeconomic factors will likely influence how workers, employers, and clinicians respond to the recommendations. In particular, the blood lead concentrations for which some major interventions, such as removal from lead exposure, are recommended are considerably lower than those explicitly specified in the current OSHA lead standards . The OSHClinical laboratories routinely offer brief interpretative guidance on the forms that report the result of blood lead concentrations. There is considerable variability among laboratories regarding the content of such guidance, and laboratories exercise their own discretion regarding the source and detail of the information they provide. Unlike the management guidance chart for childhood blood lead concentrations published by the


Two 3D surface scanners using collimated light patterns were evaluated in a new application domain: to document details of surfaces similar to the ones encountered in forensic skin pathology. Since these scanners have not been specifically designed for forensic skin pathology, we tested their performance under practical constraints in an application domain that is to be considered new.Two solid benchmark objects containing relevant features were used to compare two 3D surface scanners: the ATOS-II  and the QTSculptor . Both scanners were used to capture and process data within a limited amount of time, whereas point-and-click editing was not allowed. We conducted (a) a qualitative appreciation of setup, handling and resulting 3D data, (b) an experimental subjective evaluation of matching 3D data versus photos of benchmark object regions by a number of 12 judges who were forced to state their preference for either of the two scanners, and (c) a quantitative characterization of both 3D data sets comparing 220 single surface areas with the real benchmark objects in order to determine the recognition rate's possible dependency on feature size and geometry.The QTSculptor generated significantly better 3D data in both qualitative tests  that we had conducted, possibly because of a higher lateral point resolution; statistical evaluation (c) showed that the QTSculptor-generated data allowed the discrimination of features as little as 0.3 mm, whereas ATOS-II-generated data allowed for discrimination of features sized not smaller than 1.2 mm.It is particularly important to conduct specific benchmark tests if devices are brought into new application domains they were not specifically designed for; using a realistic test featuring forensic skin pathology features, QT Sculptor-generated data quantitatively exceeded manufacturer's specifications, whereas ATOS-II-generated data was within the limits of the manufacturer's specifications. When designing practically constrained specific tests, benchmark objects should be designed to contain features relevant for the application domain. As costs for 3D scanner hardware, software and data analysis can be hundred times as high compared to high-resolution digital photography equipment, independent user driven evaluation of such systems is paramount.Forensic pathology, Rough surfaces, Surface Scanning, Technology Assessment Courts typically rely on forensic pathologists to document injuries or skin conditions for the purpose of negotiating interpretation and legal significance. During these deliberations, newly raised hypotheses may require details not documented initially. Usually by the time a court trial opens, concerned deceased are buried or cremated and injuries of living people have healed – so the originals are never present any more. Skin surfaces featuring injuries can be fully digitized to be able to conduct a detailed re-analysis, but success relies on precise representation of injured skin.Other applications of precisely digitized surfaces in forensic pathology include the attempt to reconstructive juxtapositioning ,2, faciaHowever, most 3D surface scanners are not optimized for injured skin but for industrial or technical application domains such as industrial design, reverse engineering and rapid prototyping. Surfaces typically contain smooth and mostly straight or curved surfaces joined by edges or bends, holes, slots, pockets or grooves , which iIn contrast, forensic pathology of injured skin deals with complex, differently colored, locally highly reflective and small sized surface features that may still contain forensic relevance when smaller than 1 mm. We evaluated current 3D surface scanner technology for routine 3D skin surface documentation in forensic pathology, an application domain the scanners that we evaluated were not specifically designed for.The benchmark problem we posed was whether a 3D surface scanner could capture a whole body including skin findings as small as a needle mark  in a device setting typical for digitizing whole body surfaces within a short amount of time. We base this requirement on our observation that many forensic case re-evaluations focus on small (rather than large) findings, and are conducted at a time when a body has been cremated or buried or when wounds have been changed by surgical treatment or healing. We devised two benchmark objects to match these practical requirements.This paper reports handling, usage and 3D scan data comparison of our benchmark objects under practical constraints.Choice of scanners was reduced by eliminating models clearly not suitable for the task first: upon visiting an international industry exhibition , an exhaustive internet search and visits to representatives oft two companies for preliminary field tests, all but two surface scanners were either declared to yield or effectively yielded a strikingly insufficient performance for the benchmark problem. These two devices were subsequently tested: The ATOS-II by GOM , and the QTSculptor by Polygon Technology .Stability is a hard requirement for benchmark testing, so we used two  solid beBoth objects were selected and additionally modified with countersink drillings, boreholes, scratches, and felt pen marks so they would contain a range of challenging shape features typically encountered in forensic skin pathology such as fractal granularity or roughness, holes, scratches, highly reflective patches, shape convexity matching ears, hand or feet as well as discoloration. Typical features in forensic skin pathology include superficial or deep abrasions that may contain highly reflective regions such as body fluids or attached material such as gravel Fig. , injurieBoth scanners projected collimated white light patterns for optical triangulation and manufacturers' declared specifications did not differ significantly Table . CalibraBoth scanners require objects to be captured from different directions and for this test, objects were placed on a turning table and 8 – 12 single scans were acquired. Automatic image merging for 3D surface model generation, which was done subsequentially, required reference point stickers to be placed on objects only for the ATOS-II scanner prior to the scan but not for QTSculptor. Software control of scanner was integrated with computer hardware and subsequent scan image registration was based on particular file formats for both devices. This is why we evaluated each hardware-software package sold as "3D scanner" in conjunction.The ATOS-II used for this evaluation was owned by a local government department and operated by a professional who had completed a considerable number of scans. The QTSculptor PT-M1280 was tested by a novice on the manufacturer's premises. The nature of the benchmark test had been declared to the investigators prior to the scans.We proceeded to compare resulting 3D data without further manual point-click editing of the data which – as opposed to mathematical operations applied to whole images – could be viewed as tampering with visual evidence in forensic sciences. Relevant differences pertaining to duration of worksteps are contained in Table 3D data was surface-rendered using a non-texturized gray surface structure, oblique virtual illumination and orthogonal projection  or right (Y)  into a match of the ideal real object (μ) with additional deviating features (e) :yscanner = μobject + edeviating_features [Eq.1]We expected the deviation of the surface data from the benchmark object to be possibly dependent on the minimal extent (s) and appearance (a) of shape elements .edeviating_features = f [Eq.2]de-facto standard in forensic pathology. Any automatic method eligible as reference would have to be suitably accurate with a ratio of 10:1 over the method tested [Currently, macroscopic and microscopic inspection, including subsequent verbalization and categorization of observations, is the only technique yielding sufficient accuracy that is recognized as d tested   and a microscope-mounted  scanner camera . 3D surface data was processed and visualized using IDL  on a workstation . Statistical computations were performed using the software packages JMP  and SYSTAT .Both scanners provided a straightforward overall approach to object setup and handling. Some reference point stickers required by the ATOS-II fell off without apparent reason, and were replaced before the scan, but not during the scan procedure. Total time requiring user attendance was less than half the time on the QTSculptor scanner than on the ATOS-II scanner. ATOS-II generated considerably larger 3D models than the QTSculptor Table .Qualitatively , similarities and differences of scanner generated data can be appreciated by comparing photographs  with matching scan data of ATOS-II  and QTSculptor .While the overall appearance of both scanner's data seems to match the original object at first glance, individual surface patches exhibit discernible differences: A bone surface scratch lacks details on the ATOS-II generated surface  but is well discernible on QTSculptor derived data . The roughness of the finely granular bone surface  appears to be adequately represented on QTSculptor generated data  but not on the surface documented with ATOS-II , where a markedly less granular structure is exhibited. Slightly more coarsely granular surface  exhibits defects on ATOS-II  but not on QTSculptor . Small grooves present on the sandstone rock  are discernible on QTSculptor derived data  but not on the ATOS-II generated surface ; two distinct humps present on the rock (5a/2: arrows) can be differentiated on QTSculptor data (5c/2) but not on the data off the ATOS-II scanner (5b/2) where that distinction is blurred. Borehole rims (4a/2) are available for inspection in QTSculptor data (4c/2) but not on ATOS-II derived data (4c/2). Black patches (6b/photo) seem to cause the ATOS-II to represent a hole (6b/Y), whereas the QTSculptor contains a surface patch (6b/X). Shiny quartz inclusions  lead to defective surface data under the ATOS-II scanner  but not when scanned with QTSculptor . Furthermore, ATOS-II generated surface contains some shape information not present on the object; correlates for these are locations of reference point stickers .Overall the ATOS-II generated surfaces contain more details, better representation of highly reflective and finely granular rough surfaces, better representation of dark surface regions, and more surface attached to holes.For each of the 12 individual test sets, a total of 20 votes per test set yielded an average of 17.3 ± 2.2  in favor of QTSculptor generated data. In all 240 answers obtained by forced choice, 208 (87.0 ± 3.4%) votes were issued for QTSculptor generated data which is significantly more than the 32 (13.0 ± 3.4%) votes yielded by ATOS-II generated visual objects (bootstrapped standard deviation using 2000 re-samples of the size n = 100 (out of 240); p < 0.0001, Wilcoxon nonparametric test).2: p < 0.001) . Overall recognition rate as well as recognition rate for each of the two groups – category A > 0.7 mm and category B ≤ 0.7 mm – was significantly better for QTSculptor (χ1) Table .Logistic regression see Fig.  showed tClassification of 220 single surface areas yielded 79 bone surface areas, 5 bone surface areas with black discoloration, 2 bone surface areas with red discoloration, 127 sandstone and 7 quartz inclusion areas. The shape elements contained were 176 granular, 27 directional, 17 complex; patterns observed were 182 non-repetitive and 38 repetitive. No geometric characteristic  caused significant differences in recognition rates between both scanners tested. In particular, directional shapes (such as scratches) and granular shapes  did not yield significantly different recognition rates. The ATOS-II scanner showed difficulty in digitizing highly reflective (sandstone quartz inclusions) or discolored  surface regions.Three examples representative of the total of the 220 instances that we analyzed are shown in Fig. Realistic tissue samples showed to be unreliable for benchmarking 3D scanners as decay and plasticity cause skin to exhibit varying 3D shapes. Instead, we used two solid benchmark objects that contained representative geometric aspects of relevant forensic skin pathology  for an adequate representation of a real feature  while a Higher effective resolution of the QTSculptor is also indicated by the better representation of fine granularity, observed on digitized rough surfaces; a coarser grain contained in matching ATOS-II generated data indicates aliasing as a result of sub-Nyquist sampling frequency, i.e. undersampling or insufficient resolution in relation to the structure under study ,25. The Experimental subjective evaluation of the surface samples by 12 judges conclusively showed that 3D surface generation of the QTSculptor is significantly superior compared to the ATOS-II. It is known that subjective evaluation is fast and highly effective  and reliTechnical flaws in the surfaces obtained by the ATOS-II scanner included reference point stickers that not only created round bumps on the digitized 3D surface, but also, covered up object surface underneath. One could justify using reference stickers if they would cause the result to be of greater accuracy or if the scan process would progress significantly faster due to these stickers. Systems using point stickers might perform better in slight moving or not full stable target conditions; yet the ATOS-II scanner neither produced results of greater accuracy nor did it exceed the speed of the data capture process of the QTSculptor.An important reason for the ATOS-II scanner being outperformed by the significantly cheaper QTSculptor may be the application domain it is specifically designed for – industrial surface scanning. There, reference point sticker based methods may outperform any other 3D method in terms of accurately and precisely placing single 3D coordinates in data space while safely interpolating points in between – yet that was not the output required in this benchmark test. In fact, manufacturer's specification of what is or is not accurate may not coincide with any particular application's requirement for accuracy.Theoretical advantages of high-quality digital 3D-documentation include the option of examining complex 3D shapes from close-up at any later point in time without the limitation of depth-of-field, always producing focused 2D-imagery. This limitation of real photography cannot be overcome even by employing latest technology such as a plenoptic camera .We have shown that despite similar manufacturers' specifications, one 3D scanner (QTSculptor) significantly outperformed another model (ATOS-II) both quantitatively and qualitatively under practical constraints in a specific benchmark test that was devised for an application domain neither of the two scanners had been specifically designed for – forensic skin pathology.As costs for 3D scanner hardware, software and data analysis can be hundred times as high compared to high-resolution digital photography equipment, independent user driven evaluation of such systems is paramount.The author(s) declare that they have no competing interests.MH and WS both practically organized and supervised the benchmark testing and discussed ideas to write up the manuscript. WS planned the study, carried out the design of the test and the benchmark objects, studied relevant literature, analysed the resulting data, documented the figures, conducted the statistics and wrote the manuscript. WB and MS contributed to the content of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


When immunologists discovered in the 1970s that human immune cell receptors recognize tens of millions of antigens—telltale signs of a foreign invader—they were mystified. How could a genome with just 100,000 genes  generate this staggering diversity? The answer came with the discovery of a gene rearrangement process that draws on millions of possible permutations to create a unique surface receptor on every T and B lymphocyte—the immune system’s main weapons. But gene diversification of B lymphocytes doesn’t stop there. The immunoglobulin genes, which encode the Y-shaped antibodies that flag infectious microbes for destruction, undergo additional point (single–base pair) mutations that enhance pathogen recognition.Rad6 DNA-repair gene) steps in to either recruit specialized translesion polymerases to bypass the damage, or to copy the correct base sequence from the intact sister strand. While adept at dealing with damaged DNA, the translesion polymerases are “error-prone” compared to standard replicative DNA polymerases. This tendency can be beneficial within the immunoglobulin genes, however, because the introduced mutations can improve antigen recognition of the encoded antibodies. But it was unclear how translesion polymerases are recruited for immunoglobulin hypermutation.In a new study, Hiroshi Arakawa, Stefan Jentsch, and Jean-Marie Buerstedde find a surprising link between one of these mutation-inducing mechanisms, called immunoglobulin hypermutation, and a DNA repair pathway. When the transcription machinery encounters a DNA lesion, the RAD6 pathway (named after the Rad6 and Rad18 tag proliferating cell nuclear antigen (PCNA) with a small molecule called ubiquitin. The tag is added to a conserved lysine amino acid residue, called K164—a ubiquitination target in eukaryotes from yeast to humans. Yeast PCNA can undergo modifications at K164 by one ubiquitin tag (mono-ubiquitination), multiple tags (poly-ubiquitination), or by small, ubiquitin-related modifiers (SUMOs) in response to DNA damage. An amino acid substitution from lysine (K) to arginine (R) at position 164 (K164R) in yeast prevents the ubiquitination and SUMOylation but does not compromise the functions of the unmodified PCNA.Studies in yeast show that K164RPCNA mutation either alone or in combination with mutations that inactivated the RAD18 or REV1 genes.The role of PCNA ubiquitination has been well characterized in yeast but not in higher eukaryotes. It had been reported that human PCNA undergoes only mono-ubiquitination at K164, which increases its affinity for two translesion polymerases, Polη and REV1. Working with a chicken B cell line whose genetic tractability has made it a favored model for studying DNA repair and immunoglobulin hypermutation, the authors generated a series of clones carrying the K164RPCNA mutation showed neither modification. PCNA mono-ubiquitination was markedly reduced, but not eliminated, in cells lacking functional RAD18 genes. The K164RPCNA mutation also made cells vulnerable to DNA-damaging agents—likely because PCNA failed to recruit the translesion polymerases—suggesting that vertebrates also require PCNA ubiquitination at the K164 site to survive DNA damage.The authors analyzed extracts from the progenitor line and mutant clones to look for PCNA modifications. Mono-ubiquitinated and “SUMOylated” PCNA was evident in nonmutant cells, but clones carrying the K164RPCNA mutant clone. Loss of RAD18 or REV1, however, reduced the rates of mutations by about 2-fold and 3- to 4-fold, respectively.All the clones under study expressed immunoglobulin on their surface, allowing the authors to easily track the appearance of harmful mutations in the immunoglobulin variable regions (the targets of hypermutation). Whereas the progenitor line (serving as a control) showed a high rate of immunoglobulin loss (about 35% after 2 weeks culture), the rate was 7-fold reduced in the K164RPCNA single amino acid substitution not only renders cells sensitive to DNA damage but also dramatically impairs their capacity for immunoglobulin hypermutation. Both effects most likely result from the absence of ubiquitination, since the RAD18 mutant clone displays a similar but less severe DNA repair and hypermutation defect. Now that it’s clear that the immune system has tailored the ubiquitination-PCNA pathway to its own needs, researchers can begin to work out the molecular details of this appropriation—and perhaps explain how vertebrates managed to maximize the benefits of one pathway by using it in two systems critical for the survival of the organism.These results demonstrate that the