It is known that there is an increase in the prevalence of allergy and that allergic diseases have a negative impact on individuals' health-related quality of life (HRQL). However, research in this field is mainly focused on individuals with verified allergy, i.e. leaving out those with self-reported allergy-like conditions but with no doctor-diagnosis. Furthermore, studies on food hypersensitivity and quality of life are scarce. In order to receive information about the extent to which adolescent females and males experience allergy-like conditions and the impact of these conditions on their everyday life, the present study aimed to investigate the magnitude of self-reported allergy-like conditions in adolescence and to evaluate their HRQL. Special focus was put on food hypersensitivity as a specific allergy-like condition and on gender differences.In connection with lessons completed at the children's school, a study-specific questionnaire and the generic instrument SF-36 were distributed to 1488 adolescents, 13–21 years old (response rate 97%).Sixty-four per cent of the respondents reported some kind of allergy-like condition: 46% reported hypersensitivity to defined substances and 51% reported allergic diseases (i.e. asthma/wheezing, eczema/rash, rhino-conjunctivitis). A total of 19% reported food hypersensitivity. Females more often reported allergy-like conditions compared with males (p < 0.001). The adolescents with allergy-like conditions reported significantly lower HRQL (p < 0.001) in seven of the eight SF-36 health scales compared with adolescents without such conditions, regardless of whether the condition had been doctor-diagnosed or not. Most adolescents suffered from complex allergy-like conditions.The results indicate a need to consider the psychosocial impact of allergy-like conditions during school age. Further research is needed to elucidate the gender differences in this area. A team approach addressing better understanding of how allergy-like conditions impair the HRQL may improve the management of the adolescent's health problems, both in health-care services and in schools. An increase in the prevalence of asthma and atopy during the last two decades is documented for both children  and adulIt is well known that there are more individuals with perceived hypersensitivity than individuals with verified/doctor-diagnosed allergy. This is especially true when it comes to perceived food hypersensitivity with up to tenfold higher figures versus verified food allergy ,6. PrevaResearch has demonstrated that allergic diseases have a negative impact on individuals' health-related quality of life (HRQL) and a number of studies describe HRQL-deteriorations in children and adults with asthma -12, eczeAdverse reactions to food constitute an important part of allergy-associated problems, especially among children. Still, studies on food allergy and HRQL are scarce. However, parental perception of physical and psychosocial functioning, measured with the Children's Health Questionnaire (CHQ-PF50), has shown that childhood food allergy has a significant emotional impact on the parent and limits the family activities . It has Previous studies have shown that adolescent males have a higher prevalence of atopy than adolescent females  althoughResearch in the allergy field is mainly focused on individuals with verified allergy and their suffering from these conditions, i.e. leaving out those with self-reported allergy-like conditions but with no doctor-diagnosis. Still, one can presume that perceived allergy could have an impact on the health-related quality of life (HRQL) and involve suffering, regardless of verifiable diagnosis.In order to obtain information about the extent to which adolescent females and males themselves experience allergy-like conditions and the impact of these conditions on their everyday life, the present study aimed to investigate the magnitude of self-reported allergy-like conditions in adolescence and to evaluate their HRQL. Special focus was put on food hypersensitivity as a specific allergy-like condition and on gender differences.The present study involved adolescents at the senior level of the nine-year compulsory school and at the upper secondary school in a municipality in the south of Stockholm, Sweden. The socio-demographic distribution of the inhabitants in this municipality was slightly above in socio-economics and slightly below in number of immigrants compared with the country as a whole. A total of 2064 adolescents were registered in the schools at the relevant levels, all with the Swedish language as their school language.One week prior to starting the study (May 2003), an information letter outlining the purpose of the investigation, including an assurance of confidentiality and voluntary participation, was distributed to both the adolescents (n = 2064) and their parents. In connection with lessons at school, the teachers distributed questionnaires to the 1488 adolescents who were present at school. The instructions given by the teachers and the administration of the questionnaires were standardized. Two questionnaires were distributed together in one envelope, with the HRQL-questionnaire (se below) at the top. The adolescents themselves completed the questionnaires during that particular lesson. No other support was offered than the possibility to ask the teacher clarifying questions regarding the wording.After completion of the questionnaires, or in case of renouncing participation, each adolescent put the questionnaires into the envelope, sealed it, and handed it over to the teacher, who forwarded the envelopes to one of the authors (BM). The discrepancy between the number of registered adolescents (n = 2064) and the number, who were actually present when the data was collected (n = 1488), was partly due to the fact that it was close to the summer holiday and graduation and some adolescents attended activities outside school. The school records confirmed their absences. As 37 adolescents had not properly filled in the questionnaires, those were excluded from the study. In total 1451 questionnaires (97%) remained for data analysis. Age ranged between 13 and 21 years (mean 16.2 years), with 99% of the adolescents between 14 and 20 years. In total 696 females and 716 males had reported their gender. For 39 adolescents the gender was not reported.The terminology used in this study is according to ISAAC   and EAACA study-specific questionnaire was used to evaluate the magnitude of allergy-like conditions during the past twelve months and to evaluate the frequency of allergy testing. The questionnaire, devised by the authors, was based on relevant literature on similar subjects . Prior t"Are you allergic or hypersensitive to any of the following?" "If you are allergic or hypersensitive to any food items, what reactions or symptoms do you perceive? ."in the past 12 months? (Yes/No)""Have you had asthma, wheezing or whistling in the chest In the past 12 months, have you had recurrent eczema or itchy rash for at least 6 months? (Yes/No)""In the past 12 months, have you had a problem with sneezing, or a runny, or a blocked nose when you did not have a cold? (Yes/No)""In the past 12 months, have you had a problem with itchy-watery eyes when you did not have a cold? (Yes/No)""The questions concerning allergic diseases were taken verbatim from the ISAAC study .The generic instrument Medical Outcome Trust Short Form 36 Health Survey (SF-36) was used to measure HRQL. SF-36 is a well-validated and reliable measure of HRQL in adults and adolescents from the age of 14, and normative data are available for the Swedish population . The SF-For statistical analyses the SPSS 11.0 program was used. SF-36 data was processed by means of an SPSS program provided by the HRQL-group at the University of Gothenburg, Sweden . InternaTo test differences in proportions between groups, the Chi-square test was used. The Student's t-test and, when appropriate, the one-way analysis of variance (ANOVA) were used to assess differences in means between groups. A p-value <0.05 was considered to be statistically significant.This study was approved by the Director of School Administration in Tyresö municipality and by the research ethics committee at Huddinge University Hospital.As shown in Table The adolescents suffered to a large extent from complex allergy-like conditions, i.e. hypersensitivity to multiple offending substances and/or allergic diseases. Fifty per cent of those with hypersensitivity (n = 334/663) reported more than one kind of offending substance and 35% of those who reported allergic diseases (n = 260/739) reported more than one type of disease. Fifty-one per cent of those with allergy-like conditions (n = 471/931) reported both hypersensitivity and allergic disease.Significantly more females than males reported allergy-like conditions Table . In addiOut of the 931 adolescents with allergy-like conditions, 404 (43%) reported that they had been tested for allergy by means of blood test or skin prick test (results not shown). For the majority (n = 324) of these adolescents with self-reported allergy testing, the tests had been performed during their school age years. Sixty-one per cent of those tested (n = 246/404) reported that the test results verified allergy. This figure corresponds to a 17% prevalence of self-reported verified allergy within the whole population of 1451 adolescents.In the group of 271 adolescents reporting food hypersensitivity, 139 (51%) reported allergy test results that verified some kind of allergy, albeit not necessarily food allergy (results not shown).The most common food-induced symptoms were OAS -like symptoms Table , i.e. itOffending food items reported were: nuts (39%), fruit and berries (35%), peanut (32%), almond (22%), tomato (19%), carrot (16%), lactose (12%), vegetables (10%), shellfish (9%), soy (7%), milk (7%), fish (5%) and egg (5%). Substances reported for less than five per cent of the 271 adolescents are not specified. For two food items there were significant gender differences. The offending food items fruit and berries were more commonly reported by females (44% and 24% respectively, p < 0.001) and peanut was more commonly reported by males (43% and 27% respectively, p < 0.01).A total of 63 adolescents reported food as the only offending substance. However, the majority of the 271 adolescents who reported food hypersensitivity suffered from complex allergy-like conditions that included additional offending substances besides food (77%) as well as allergic diseases (78%). In this group of food hypersensitive adolescents there were significantly more males than females who reported hypersensitivity to furred animals and as regards hypersensitivity to nickel the result was reverse  (n = 210/271) to those food hypersensitive adolescents who did not report such conditions (n = 61/271), the groups showed a similar pattern as regards the mental dimension scales of the SF-36, i.e. no statistically significant differences were found. As regards the physical dimension scales, the food hypersensitive adolescents who also reported allergic diseases scored significantly lower on BP (bodily pain) and GH  Figure .A comparison within the whole group of adolescents with allergy-like conditions n = 931), i.e. between those who reported only hypersensitivity to any defined environmental substances (A), those who reported only allergic diseases (B), and those who reported both (C), showed that the three groups scored similar on the four health scales representing the mental dimension of the SF-36. As regards the physical dimension, adolescents with allergic diseases only (B) or in combination with hypersensitivity (C) scored lower on the health scales for bodily pain (BP) and general health (GH) compared with those with only hypersensitivity to defined substances (A)  showed no significant difference. This is noteworthy, as the physical parameter often is in focus when health-care professionals assess an individual's state of health. However, it has been previously shown that in patients with asthma/wheezing, the link between lung function and HRQL is weak ,12 and tIn the present study we show that in adolescence, significantly more females than males experienced not just asthma/wheezing but also eczema/rash, rhino-conjunctivitis, and hypersensitivity to food, dust/mite and nickel. The females presented more complex allergy-like conditions compared with the males. In addition, females with allergy-like conditions showed more severe HRQL-deterioration compared with the males. It is known that female gender among adults implies a larger report of burden of health problems in general  and SF-3Seventeen per cent of the adolescents, of whom the most part were tested during school age, reported positive allergy test results. It can be assumed that there were some additional adolescents with doctor-diagnosed allergy but without verifying test results. Thus, the total of adolescents doctor-diagnosed as having allergy may well be more than 17%. However, in the present study the focus was on self-reported (perceived or verified) allergy-like conditions. The mean SF-36 scores did not differ whether the adolescents reported objectively verified allergy or not. The lack of difference in HRQL was not surprising as a diagnostic test that verifies allergy says nothing about the individuals experience or the severity of the allergic condition.Most food allergies are something that children outgrow  and adveFood items constituted a considerable part of the offending substances reported in this study and up to 41% of all adolescents with hypersensitivity specified at least one food item as an offending substance. Professional counselling and diagnostic procedures may to some extent be able to help the adolescents to reduce their food avoidance. Yet, this kind of perceived allergy-like condition – regardless of what the underlying mechanisms were – was evidently associated with HRQL-deterioration and the adolescents' experiences deserve sincere attention. Previous research show that food allergy in a child implies disruption in daily life and HRQL-deterioration for both the child and the family -21.A pattern emerged in this group of food hypersensitive adolescents, showing a great deal of hypersensitivity to pollen, rhino-conjunctivitis and OAS-like symptoms, which is in accordance with the well known cross-reactivity of pollen and food allergens . SignifiMore than half of the adolescents with food hypersensitivity reported positive allergy tests, but as a consequence of how the questions in the survey were asked, it is not known if they were verified as allergic specifically to food. However, a vast majority of the food hypersensitive adolescents did undoubtedly suffer from complex allergy or allergy-like conditions, which included multiple types of hypersensitivity and/or allergic diseases. This should require competence in health-care when trying to tackle the adolescents' multifaceted problems, including food hypersensitivity.The extensive number of adolescents who participated in the present survey, together with a very high answer rate, makes the results reliable. However, the sample of adolescents used in this study may limit the generalizability as the socio-demographic distribution in this particular municipality was slightly above in socio-economics and slightly below in number of immigrants compared with the country as a whole. Additional studies are warranted to confirm the results.The results of the present study emanate exclusively from adolescents' statements. There is always a risk that the respondents involved do not correctly remember things that were asked for in a questionnaire. However, our main interest was in adolescents' experiences of allergy-like conditions during the past twelve months and daily functioning during the past four weeks. Remembering correctly over a longer period of time was only of importance in questions about allergy testing. It seems likely that the adolescents would remember such events as skin prick tests or blood tests during their school age, although tests carried out in early childhood might have been unknown or forgotten.The magnitude of allergic diseases was measured by means of ISAAC-questions. The ISAAC questionnaire, which asks for events during the past 12 months, has been used in many countries all over the world and for many years. Its results constitute basis for international comparisons and it can be considered well validated. However, hypersensitivity items are not included in the ISAAC questionnaire. The questions in the present study about hypersensitivity were developed specifically for the present study and a pilot test was performed. Only lexical adjustments were needed.It could be discussed if the fact that the questionnaires asked for events during two distinct periods: 12 months  and 4 weeks (daily functioning/SF-36) might have biased the present results. It is also possible that an allergy-specific HRQL measure instrument would give another picture of the physical scale in relation to allergy-like conditions.The prevalence of self-reported allergy-like conditions among adolescents was high – 64%. Significantly more females than males reported allergy-like conditions and females with allergy-like conditions showed more severe HRQL-deterioration compared with males with such conditions. The results indicate a need to consider not merely physical consequences but also the psychosocial quality of life impact of allergy-like conditions among both females and males. Further research is needed to elucidate the reasons behind the gender differences in this area.Most adolescents suffered from complex allergy-like conditions that included multiple types of hypersensitivity and/or allergic diseases. Food items constituted a considerable part of the offending substances reported. When attending to a young individual who suffers from an allergy-like condition, the whole syndrome should be in focus – not only one specific offending substance, or one specific hypersensitivity or allergic disease. A team approach accompanied by an understanding of how allergy-like conditions impair the quality of life may improve the management of the adolescent's health problems, both in health-care services and in schools.BM and GN conceived of the study. All authors made substantial contributions to conception, planning and design. BM carried out the acquisition, analysis and interpretation of data. BM drafted the manuscript. GN and SA have been involved in revising it critically for important intellectual content. All authors read and approved the final manuscript.


The identification of disease-associated genes using single nucleotide polymorphisms (SNPs) has been increasingly reported. In particular, the Affymetrix Mapping 10 K SNP microarray platform uses one PCR primer to amplify the DNA samples and determine the genotype of more than 10,000 SNPs in the human genome. This provides the opportunity for large scale, rapid and cost-effective genotyping assays for linkage analysis. However, the analysis of such datasets is nontrivial because of the large number of markers, and visualizing the linkage scores in the context of genome maps remains less automated using the current linkage analysis software packages. For example, the haplotyping results are commonly represented in the text format.Here we report the development of a novel software tool called CompareLinkage for automated formatting of the Affymetrix Mapping 10 K genotype data into the "Linkage" format and the subsequent analysis with multi-point linkage software programs such as Merlin and Allegro. The new software has the ability to visualize the results for all these programs in dChip in the context of genome annotations and cytoband information. In addition we implemented a variant of the Lander-Green algorithm in the dChipLinkage module of dChip software (V1.3) to perform parametric linkage analysis and haplotyping of SNP array data. These functions are integrated with the existing modules of dChip to visualize SNP genotype data together with LOD score curves. We have analyzed three families with recessive and dominant diseases using the new software programs and the comparison results are presented and discussed.The CompareLinkage and dChipLinkage software packages are freely available. They provide the visualization tools for high-density oligonucleotide SNP array data, as well as the automated functions for formatting SNP array data for the linkage analysis programs Merlin and Allegro and calling these programs for linkage analysis. The results can be visualized in dChip in the context of genes and cytobands. In addition, a variant of the Lander-Green algorithm is provided that allows parametric linkage analysis and haplotyping. The oligonucleotide Mapping 10 K arrays -4 and thHere we report the development of a new software tool called CompareLinkage that can be used for automated conversion of Mapping 10 K genotype data into the "Linkage" format for linkage analysis in Merlin, GeneHunter and Allegro . In addiTo analyze large pedigrees rapidly and to compare the linkage analysis results of different software packages, we developed a software tool called CompareLinkage to automate the following processes: (1) Converting of Affymetrix Mapping 10 K genotype data, pedigree files and marker information into the "Linkage" format , and detA graphical user interface (GUI) for Windows was also implemented in Java. In this GUI users are allowed to set their own working directory and the location of the Perl interpreter through the "Setting" menu. CompareLinkage's functions of converting file formats and getting dChip input files are incorporated through the "Convert" and "GetCurve" menu Figure . Since cThe Affymetrix Mapping 10 K array CEL files and genotype TXT files can be imported into dChip and visualized along cytobands and genes as previously reported ,11. The v involves the summation over all the possible real genotypes :We implemented a variant of the Lander-Green ,6,16 algi represents the ith of all the possible founder allele configurations and is independent of v. P is 1 since an inheritance vector and founder allele configuration uniquely determines the real genotypes, and P involves comparing the real genotype and observed genotype for all the individuals and multiplying the probability by the error rate of 0.01  for each disagreement and 0.99 for each agreement. We also use the matrix-vector multiplication algorithm and bit reduction due to founder phase symmetry described in [where Fribed in , and theribed in ,17 to spWe use the forward-backward computation in the Lander-Green algorithm to obtain the marginal probability distribution of inheritance vector at each SNP marker position given the data of all the markers on a chromosome. In addition the most likely inheritance vector at each marker given the genotype data of all the markers on this chromosome is calculated . ConditiCompareLinkage can format Affymetrix Mapping 10 K SNP genotype output files and genotype files into the "Linkage" format and convert genome information and pedigree files into the formats suitable for Merlin (Version 0.10.2), GeneHunter (Version 2.1) and Allegro (Version 1.2). CompareLinkage removes all non-informative markers and calls the PedCheck software  to detecFigures To do parametric linkage analysis in dChipLinkage, a pedigree file is needed Figure . The fil1. Open dChip.Analysis menu and the Get External Data function to read in the genotype file in the text format  with LOD scores of greater than 2.3 Figure  and the After the linkage computation is finished, the inferred haplotype information can be visualized. In the haplotype view Figure  and 16, We have developed the CompareLinkage software for easy comparison and analysis of genotype datasets with common multi-point linkage analysis software programs. It provides functions such as automated data formatting and the calling of linkage analysis software programs to facilitate comparative linkage analysis. The results can be visualized in a chromosome window in the context of genes, cytobands and SNPs in dChip's user friendly graphical interface. The linkage scores of other linkage software packages can be saved into the dChip score file format through CompareLinkage and viewed in the dChip chromosome viewer. This provides the interface to view other computed statistics such as linkage disequilibrium scores along the chromosomes. We have also implemented a variant of the Lander-Green algorithm as the dChipLinkage module for parametric linkage analysis of small pedigrees. It can analyze all chromosomes for families with up to 18 bits within one hour on a PC with one gigabyte memory. This is useful for recessive and consanguineous families whose bits are often small.The comparison analysis of three Mapping 10 K array data sets show similar results in regions with significant LOD scores across all the four software packages. The regions with concordant LOD/NPL scores should provide more confidence in the candidate disease loci. However, there are clear differences in isolated regions. This emphasizes the challenge of a comparative analysis using different linkage algorithm implementations. We hypothesize that the differences between the software programs in peak locations are attributable to:1. The specific algorithm implementation in each program.2. The difference between parametric – and non-parametric analysis.3. The existence of undetected genotype errors in the data sets which could falsely deflate LOD scores ,22. dChiIn light of the discordance between the results from common linkage software packages and from dChipLinkage, we will validate dChipLinkage implementation using additional datasets and the CompareLinkage software.In summary, the CompareLinkage and dChipLinkage software automate the comparative linkage analysis and visualization using multiple software packages. With these tools users will be able to increase their confidence in candidate regions and can use the visualization tools to explore the disease associated genome regions.Project name: The CompareLinkage software and the dChipLinkage software moduleProject home page: Operating system(s): Windows (dChipLinkge); Windows , Unix (CompareLinkage command line version)Programming language: Visual C++ 6.0 (dChipLinkge); Perl and Java (CompareLinkage software)Other requirements: NoneLicense: None.Any restrictions to use by non-academics: No restrictionsCR, CL and WHW conceived of the study, and participated in its design and coordination. NM and RJHS generated the 5026.10 family data, and MP generated the CR and ER family data. IL implemented the CompareLinkage software and performed the comparative analysis using multiple linkage analysis software packages. JC implemented its graphical user interface (GUI). CL implemented the dChipLinkage module. KH participated in the design and analysis of the study. IL, CR and CL drafted the manuscript. All authors read and approved the final manuscript.


Acetylcholine receptor type ligand-gated ion channels are well known in animals. Homologs are identified in prokaryotes that may act as chemotactic receptors. 2+ ions. They play a central role in fast synaptic signaling in animal nervous systems and so far have not been found outside of the Metazoa.Acetylcholine receptor type ligand-gated ion channels  are a superfamily of proteins that include the receptors for major neurotransmitters such as acetylcholine, serotonin, glycine, GABA, glutamate and histamine, and for ZnMethanosarcina. The homology between the animal receptors and the prokaryotic homologs spans the entire length of the former, including both the ligand-binding and channel-forming transmembrane domains. A sequence-structure analysis using the structure of Lymnaea stagnalis acetylcholine-binding protein and the newly detected prokaryotic versions indicates the presence of at least one aromatic residue in the ligand-binding boxes of almost all representatives of the superfamily. Investigation of the domain architectures of the bacterial forms shows that they may often show fusions with other small-molecule-binding domains, such as the periplasmic binding protein superfamily I (PBP-I), Cache and MCP-N domains. Some of the bacterial forms also occur in predicted operons with the genes of the PBP-II superfamily and the Cache domains. Analysis of phyletic patterns suggests that the ART-LGICs are currently absent in all other eukaryotic lineages except animals. Moreover, phylogenetic analysis and conserved sequence motifs also suggest that a subset of the bacterial forms is closer to the metazoan forms.Using sensitive sequence-profile searches we have identified homologs of ART-LGICs in several bacteria and a single archaeal genus, From the information from the bacterial forms we infer that cation-pi or hydrophobic interactions with the ligand are likely to be a pervasive feature of the entire superfamily, even though the individual residues involved in the process may vary. The conservation pattern in the channel-forming transmembrane domains also suggests similar channel-gating mechanisms in the prokaryotic versions. From the distribution of charged residues in the prokaryotic M2 transmembrane segments, we expect that there will be examples of both cation and anion selectivity within the prokaryotic members. Contextual connections suggest that the prokaryotic forms may function as chemotactic receptors for low molecular weight solutes. The phyletic patterns and phylogenetic relationships suggest the possibility that the metazoan receptors emerged through an early lateral transfer from a prokaryotic source, before the divergence of extant metazoan lineages. The flux of ions across excitable cellular membranes is a signaling mechanism that is extensively utilized by organisms from all the three major superkingdoms of life. This directional flow of ions across cellular membranes is mediated by a wide range of ion channels that may be gated by a variety of signals, such as voltage, mechanical forces or chemical first messengers . Ion-depAll the known members of this superfamily possess stereotypic domain architectures, with an all-β amino-terminal ligand-binding domain (LBD) and a carboxy-terminal transmembrane domain comprised of four membrane-spanning helices 4-TM). The members of this superfamily exhibit a pentameric quaternary structure, with the second transmembrane helix from each monomer (helix M2) contributing to the wall of a transmembrane pore through which the ion passes. The animal ART-LGICs may exist as heteropentamers, containing up to four distinct paralogous monomers. The ligand is bound at the dimer interface of two adjacent LBDs, and residues from both subunits form a box-like cavity to accommodate the ligand ,10. In t-TM. The Caenorhabditis elegans MOD-1), allow the flow of anions. Cation or anion selectivity of the channel is principally governed by the charge distribution in the linker between the transmembrane helices M1 and M2 [The ART-LGICs characterized to date show ion selectivity. The excitatory channels, such as the acetylcholine and serotonin receptors, the mammalian Zn receptors and some invertebrate GABA receptors, allow the flow of cations, whereas the inhibitory receptors, such as those for glycine and GABA, invertebrate glutamate and histamine receptors, and some invertebrate serotonin receptors  from the snail tagnalis  and the tagnalis  have thrtagnalis . This caThus far, the ART-LGIC superfamily is known only from multicellular animals (metazoans). Phylogenetic analysis suggests that the common ancestor of the bilateral animals already possessed multiple members belonging to two major families of the superfamily that correspond, respectively, to the excitatory cationic channels, including the acetylcholine and serotonin receptors, and the inhibitory anionic channels, including the GABA, glycine and invertebrate histamine and glutamate receptors ,14,15. TWe report here the identification of several prokaryotic members of the ART-LGIC superfamily and discuss the general implications of these proteins for the mechanisms and origin of the Cys-loop receptors of the animal nervous system.bona fide animal members of this superfamily (with the exception of snail ACHB) contain a globular, extracellular, amino-terminal LBD and a carboxy-terminal 4-TM domain. The membrane-spanning helices, being compositionally biased, tend to frequently recover false positives in iterative sequence profile searches. Accordingly, we only used the globular extracellular domains of the known ART-LGIC receptors, which are typically around 200-220 amino acids in length, for our iterative sequence profile searches with the PSI-BLAST program.To investigate the origins of the animal ART-LGIC superfamily, we tried to obtain a complete picture of their phyletic spread in all organisms with currently available genomic sequence information. All C. elegans MOD-1 receptor  or the human GABA receptor α4 chain  recovered a consistent set of receptors from diverse animals with significant expect (e)-values prior to convergence (run with inclusion threshold of 0.01). Interestingly, in addition to the animal sequences these searches also recovered sequences from different bacteria. For example a search initiated with the above-mentioned acetylcholine receptors as the seed recovered Gloeobacter violaceus, Crocosphaera watsonii (both cyanobacteria) in iteration 3  and Rhodopseudomonas palustris (α-proteobacteria) in iteration 6 . However, no significant hits belonging to any of the other eukaryotic lineages, such as the fungi, Dictyostelium, plants, alveolates or Giardia were detectable. To further investigate the occurrence of ART-LGIC homologs in bacteria, we constructed a PSI-BLAST profile of the LBDs recovered in the above searches and used it to systematically search all the bacterial genomes, which are available as whole-genome shotgun reads or as completely assembled chromosomes. As a result of these searches we recovered statistically significant hits to the ART-LGIC LBDs from several other phylogenetically diverse bacteria including Cytophaga hutchinsonii, α-proteobacteria like Bradyrhizobium japonicum and Magnetospirillum magnetotaticum, γ-proteobacteria, like Erwinia chrysanthemi, Microbulbifer degradans and Methylococcus capsulatus, several cyanobacteria and a single archaeal genus Methanosarcina.Iterative searches from a number of starting queries, such as the human acetylcholine receptor α7 chain , -17 in iterations 1-3). For example, a search with the sequence of Chut0841 (gi: 23135736) from C. hutchinsonii recovered the animal receptors with e-values in the range 10-4-10-6 in the second iteration. The secondary structure was predicted for the region corresponding to the LBD in the bacterial proteins using the programs PHD [All these bacterial hits corresponded to the full length of the animal LBDs, which were used as seeds to build the sequence profiles. When signal peptides and the transmembrane helices were predicted for the bacterial proteins, all of them showed a general structure similar to the animal receptors; that is, an amino-terminal signal peptide and a LBD followed by a carboxy-terminal 4-TM domain. However, some of the bacterial proteins showed additional domains between the amino-terminal signal peptide and the ART-LGIC superfamily ligand-binding and channel domains (see below for further discussion). Reciprocal searches with either just the region corresponding to the LBD or the whole unit comprising both the LBD and the following 4-TM domain of the bacterial proteins recovered the animal Cys-loop proteins with significant e-values , which is predicted to be critical for the flexibility of the structure to conformational change [T. californica ACHR α-chain) that initiates a bend in the helix resulting in the hydrophobic constriction or girdle that forms the channel gate [T. californica ACHR α-chain, in the loop between helices M2 and M3, has been implicated as one the residues that may be critical for the rotational freedom of the ACHR M2 helix during the gating process [Over and above the general conservation of hydrophobic residues in the 4-TM domain, there are some potential functionally relevant conserved positions shared by the bacterial and metazoan proteins. One of these is the helix-bending position in helix M1 , glycine (G) or serine (S), and corresponding to P221 in l change ,28. Anotnel gate . Glycine process . The strOne of the major determinants of ion selectivity is the sequence just amino-terminal to the helix M2 on the cytoplasmic side. The cation channels usually have a sequence motif of the form glutamate (E)-[arginine (R)/lysine (K)] with the glutamate playing a role in cation selection. The anion channels usually have a motif of the form alanine (A)-[RK] with the basic residue participating in anion selection ,12,29. AThe ligand-binding box in ACHB has been termed the aromatic box as it is bounded by multiple aromatic residues Figures , 2. In sFurthermore, an interesting difference is noted in the aromaticity of the positions corresponding to leucine (L) 112 (subunit D) and tryptophan (W) 143 (subunit C) of the ACHB structure between the bacterial and animal sequences  in the middle of the region corresponding to the Cys-loop Figure  and thesThe conservation of certain key features in both the LBD and the 4-TM domains of the bacterial and eukaryotic receptors suggests that despite their extensive sequence divergence they are likely to share general functional and mechanistic properties. In the pentamer these residues appear to form a continuous ring passing through the top surface of the LBD, and undergo conformational changes in relation the presence of a bound ligand Figure  9]..9].Rhodopseudomonas and one of the three versions from C. hutchinsonii show a simple architecture identical to the animal forms. Some versions, like those from the α-proteobacteria, M. magnetotacticum and B. japonicum, show a further amino-terminal fusion to a domain of the periplasmic binding protein type I (PBP-I) superfamily  and Cache domains . This ver Figure . The MCPRhodopseudomonas, there is a similar predicted operon, but instead of a gene for a PBP-II superfamily protein, there is one for a stand-alone Cache domain. This situation parallels the fusion with the Cache domain in some of the receptor versions and these two independent proteins may similarly cooperate functionally.Contextual information in the form of conserved gene neighborhoods or predicted operons in prokaryotes often provides hints to identify gene products that functionally or physically interact or belong to the same pathways or signaling cascades ,44. Acco+ channel in the bacterium Bacillus pseudofirmus in chemotaxis, motility and the regulation of the Na+-cycle [Microbulbifer degradans, the ART-LGIC with a predicted cation selectivity is in a predicted operon with a Na+/H+ symporter, suggesting possible interactions with the Na+ cycle.Taken together, these observations suggest that bacterial ART-LGICs may function as chemotaxis receptors. As most bacterial genomes in which they are present contain only a single member of the ART-LGIC superfamily, it is likely that, in contrast to many of the well studied metazoan receptors, they function as homopentamers. The PBP-I, PBP-II MCP-N and Cache domains that are either fused or operonic with many of the predicted bacterial receptors may help in a preliminary concentration or sensing of amino acids or other small-molecule ligands. These ligands may then bind to the channel's LBD domain and activate an ionic flux across the cell membrane that in turn regulates the motility of the bacterium in response to the ligand. This proposal is analogous to the recently reported activity of a voltage-gated Naa+-cycle . InteresDictyostelium, Entamoeba, apicomplexans or earlier-branching eukaryotic taxa such as Giardia and Trichomonas. Among the prokaryotes, too, they show a highly sporadic distribution: very distantly related taxa may possess similar receptors  are markedly more similar in sequence to the eukaryotic forms . The NO receptors of animals share two domains, namely the HNOB and HNOBA, which are involved in heme-dependent NO sensing with several bacterial signaling proteins [Dictyostelium . The pattern of the residues conserved in both the metazoan and bacterial receptors suggests that a common mechanism of channel-gating is likely to operate throughout this superfamily. Furthermore, the ligand-binding box appears to preserve at least one aromatic residue, although its exact position may not necessarily be conserved. The conservation pattern also suggests that a chain of positions leading out on either side from the ligand-binding box may mediate the transmission of the conformational change through the 'top' of the LBD, which may then transmit through the rest of the structure. The charge interactions between the acidic residue in the middle of the Cys-loop region and a basic residue the extreme carboxyl terminus of the LBD, just before the transmembrane domain also appear be universal features that might be involved in the process of channel gating. On the basis of the domain architectures and operon organizations, we predict that the bacterial ART-LGICs are likely to function as chemoreceptors for low-molecular-weight solutes in the environment. Phyletic and phylogenetic analyses suggest that the ancestor of the animal lineage probably acquired a single progenitor from a bacterial source, and it subsequently radiated to give rise to all the Cys-loop receptor subunits of the extant metazoans.The nonredundant (NR) database of protein sequences ) was searched using the BLASTP program . UnfinisProtein structure manipulations were performed using the Swiss-PDB viewer program . ProteinPhylogenetic analysis was carried out using the maximum-likelihood, neighbor-joining, Bayesian inference and minimum evolution (least squares) methods. The MrBayes program was used for the Bayesian inference of phylogeny . The aliThe minimal evolution trees were constructed using the FITCH program  of the PThe following additional data are available with the online version of this paper. Additional data file The conservation pattern of the ART-LGIC superfamily plotted onto the three-dimensional structure of the ACHB proteinClick here for additional data fileThe alignment of the proteins in Figure 1Click here for additional data file


Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs) may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system.2 values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1–9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and minisequencing in a microtiter plate format.The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by RWe conclude that microarray based minisequencing is an accurate and accessible tool for multiplexed screening for imbalanced allelic expression in multiple samples and tissues in parallel. Single nucleotide polymorphisms (SNPs) are highly abundant in the human genome, appearing on average at 0.1% of the nucleotide positions -6. ImbalNon-synonymous SNPs in coding regions of genes may be functional by altering an amino acid, which in turn may affect the structure and function of the encoded protein, while synonymous SNPs may have functional consequences by affecting the stability or folding of mRNA transcripts. Intronic SNPs may give rise to alternatively spliced mRNAs, while SNPs in 5'- or 3'-untranslated mRNA regions may affect the stability or processing of the RNA. Moreover, SNPs in non-protein coding regions of genes that affect binding of regulatory factors may cause imbalanced expression of SNP alleles. This form of genetic variation has been suggested as a common cause of both normal and disease-related inter-individual variation in complex phenotypes . ClearlyOwing to the high sequence specificity of nucleotide incorporation by DNA-polymerases, single nucleotide primer extension has proven to allow quantitative determination of SNPs in genomic DNA in several studies and assay formats (for a review, see Syvänen 2001 ). A freqWe are currently using microarray based minisequencing for multiplex genotyping of SNPs. Our custom-made microarrays permit the genotyping of up to 100 SNPs in 80 samples per standard microscope slide, either using immobilised minisequencing primers ,18 or usWe used a panel of ten coding SNPs in five genes to choose the optimal microarray based minisequencing strategy for multiplex, quantitative genotyping of SNPs in DNA and RNA samples. The selected SNPs were located in genes shown by reverse transcriptase PCR analysis to be expressed in one or both of two endothelial cells lines, HUVEC  and HAEC  that served as our model cell lines in this study (data not shown). We evaluated two formats of microarray based minisequencing by performing five parallel assays with each method for each sample in the evaluation. The SNPs were analysed in both DNA polarities and the evaluation of the methods was based on the DNA polarity yielding the highest signal-to-noise ratio.In Method I, immobilised minisequencing primers are extended with fluorescently labelled ddNTPs in reactions performed on the microarray surface after annealing of the multiplex PCR products to the primers ,21. In M2), which describes how well the regression line fits the data points, was used to assess the accuracy of quantification of the SNP alleles by Methods I and II. As can be seen in Table 2 values are close to one for most of the SNPs analysed, demonstrating little scatter of the data points around the regression line. For Method I, six of the ten SNPs analysed have R2 values ≥ 0.95, while for Method II the R2 values are ≥ 0.95 for eight of the SNPs. Thus, accurate quantification of SNP alleles is possible by both methods. The slopes of the regression lines vary between the ten SNPs as well as between the two methods  and endothelin receptor type B (EDNRB rs5351) were heterozygous in the HUVEC cell line. These SNPs were genotyped in cDNA produced from total RNA extracted from the cells with the corresponding gDNA as reference samples using both methods. Table Next, the performance of the two methods in quantitative analysis on the RNA level was assessed. The ten SNPs were first genotyped in genomic DNA (gDNA) from the HUVEC and HAEC cells to identify those SNPs that were heterozygous in either or both cell lines. Three SNPs in the low density lipoprotein receptor gene  were heterozygous in the HAEC cell line, and one SNP in each of the genes encoding angiotensin I converting enzyme (ACE rs4331), βTo test that the results on imbalanced allelic expression detected by the multiplexed microarray based methods represents the true biological situation in the cells, we analysed the heterozygous SNPs in five replicate RNA samples prepared from HUVEC or HAEC harvested at different time points from different cell culture flasks. We also analysed the three LDLR SNPs in five replicate reverse transcription reactions from the same RNA sample prepared from HAEC cells. For this analysis we used our first generation solid-phase minisequencing assay for individual SNPs in a microtiter plate format. The concordant cDNA/gDNA ratios from these control experiments from independent cell and RNA samples presented in Table The purpose of our study was to evaluate microarray based minisequencing for multiplexed detection and quantification of imbalanced expression of SNP alleles, as a prelude to further large scale screening for allelic imbalance. We found no significant differences in the performance of our two "in house" methods, minisequencing with primers directly immobilised on the microarrays (Method I) and the It is notable that the largest differences in accuracy and sensitivity were observed between SNPs. Some of the SNP-to-SNP differences are likely due to differences is the accuracy and efficiency of incorporation of the four different fluorescently labelled nucleotide analogues by the DNA polymerase ,26 as weComparison of the relative amounts of the alleles of six SNPs on the RNA (cDNA) level to heterozygote SNPs in genomic DNA revealed four SNPs with imbalanced expression of the two alleles. A three-fold increase in the expression of the T-allele for the SNP rs4331 ACE was the most pronounced difference observed. In our study, 1.4–1.5-fold differences in allelic expression levels were detectable. The sensitivity of detecting a minority allele in our system would allow the distinction between 10-fold reduction in the expression of an allele and monoallelic expression, for example as a result of imprinting. Owing to its potential for high throughput screening of large numbers of samples, we have also performed a preliminary evaluation of the commercial SNPstream genotyping system (GenomeLab, Beckman Coulter) that also utilises the "tag-array" primer extension strategy in a semi-automated 384-well microtiter plate format for detection of imbalanced allelic expression . The samIt is also reassuring for future large scale detection of imbalanced allelic expression that the accuracy of our methods seemed to be similar for cDNA and genomic DNA. Analysis of replicate RNA samples from different batches of both cell lines using a microtiter plate format of the minisequencing method evidenced for the biological authenticity of the allelic imbalance detected using minisequencing in the microarray format. The data obtained from independent cell samples also indicate an acceptable reproducibility of RNA extraction, RNA storage and cDNA synthesis. Another important factor besides sample to sample variation that may affect the accuracy of the relative allele quantification is the amount of mRNA subjected to the analysis. At a low copy number of mRNA, the stochastic distribution of the RNA templates may be a major source of variation . The reaA similar minisequencing strategy as the one used for determination of imbalanced expression between SNP alleles can also be used for determination of the relative expression levels of highly homologous genes  and for Here we demonstrated the applicability of two formats of microarray based minisequencing for detecting imbalanced expression of SNP alleles. The accuracy and sensitivity of both systems allow detection of 1.4- to 10-fold differences in the expression levels of the two alleles of heterozygous SNPs. The microarray-based minisequencing systems utilise widely available reagents and equipment, and can thus easily be established "in-house". Moreover, the system is flexible with respect to number of SNPs and samples to be analyzed. Systematic quantitative screening of genetic diversity on the RNA level in multiple individuals and tissues will be a future approach in the elucidation of the molecular mechanisms that regulate gene expression.DNA samples from 30 volunteer donors were genotyped by Methods I and II to identify individuals of different genotypes for the panel of ten SNPs analysed. The SNPs are described in the section "SNPs and primers" below. DNA 10 ng/μl) from one individual was serially diluted 2:1 into DNA (10 ng/μl) from a second individual, to yield a series of DNA samples with different ratios between the SNP alleles. These mixed DNA samples were used for construction of quantification standard curves. Depending on the genotype of each SNP in the two individuals whose DNA was mixed, dilution series of samples with different allelic ranges were obtained for the ten SNPs, as specified in Table 0 ng/μl f2. Cells from the cultures were harvested at 80% confluence according to the manufacturer's instructions. Total RNA was isolated from the cells using the TRIZOL®Reagent (GIBCO BRL, Paisley, Scotland) and the RNA samples were stored at -70°C until use. High quality RNA with A260/A280 ratio over 1.9 and intact ribosomal 28S and 18S RNA were used for cDNA synthesis. The RNA samples were treated with 1 U RQ1 RNase-free DNase (Promega, Madison, WI, USA) per μg RNA. Two to 2.5 μg total RNA was subjected to first strand cDNA synthesis using SuperScript™ II (RNase H- Reverse Transcriptase, Invitrogen, Carlsbad, CA, USA) reagents in a 20 μl volume. DNA was extracted from the cells using GenElute™ Mammalian Genomic DNA Kit (Sigma, St Louis, MO, USA) and stored at -20°C until use.Human Umbilical Vein Endothelial Cells (HUVEC) and Human Aortic Endothelial Cells (HAEC) (Cascade Biologics, Inc., Portland, OR, USA) were grown in Medium 200 with Low Serum Growth Supplement (LSGS Kit, Cascade Biologics, Inc., Portland, OR, USA) at 37°C in a humidified atmosphere of 5% CO® Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), 1.5 mM MgCl2, and 0.2–0.3 μM of primers in 50 μl of 10 mM Tris-HCl pH 8.3 and 50 mM KCl. The PCR conditions were initial activation of the enzyme at 95°C for 10 min followed by 35 cycles of 95°C for 1 min, 56°C for 1 min and 72°C for 1 min and a final extension at 72°C for 7 min in a Thermal Cycler PTC225 (MJ Research, Watertown, MA, USA). The amplified fragments were combined and concentrated to 60 μl using Microcon ® YM-30 Centrifugal Filter Devices (Millipore Corporation, Bedford, MA, USA).The fragments comprising the SNPs were PCR-amplified in individual reactions using 10–15 ng genomic DNA or one tenth of the cDNA products, 0.2 mM dNTPs, 1U AmpliTaq Ten SNPs located in coding regions of genes known to be expressed in HUVEC and HAEC cells were analysed. Information on the SNPs, including dbSNP  ID numbe2-group in their 5'- or 3'-end, respectively. The oligonucleotides were applied in duplicates to the slides at a concentration of 25 μM in 150 mM sodium phosphate pH 8.5 using a ProSys 5510A instrument (Cartesian Technologies Inc, Irvine. CA, USA) equipped with one Stealth Micro Spotting pin  to minimise the variation between spots in different "subarrays". The oligonucleotides were spotted in an "array-of-arrays" configuration that facilitates analysis of 80 individual samples in parallel on each microscope slide [The minisequencing primers or the complementary tag-oligonucleotides were covalently immobilised on CodeLink™ Activated Slides  by the mediation of a NHpe slide . In eachAliquots of 7.5 μl of the concentrated PCR products were analysed in five parallel "subarrays" for each sample, essentially as described previously . The PCRFive parallel reactions with a 4.5 μl aliquot of the concentrated PCR products were analysed for each sample, as described in detail in . Excess ® DNA polymerase (Applied Biosystems, Foster City, CA, USA) and the minisequencing primer was added. The extension reaction was allowed to proceed for 10 min at 50°C. The extended primers were released with alkali and the amount of incorporated tritium labelled nucleotide was measured.PCR was run with one of the primers biotinylated. The biotinylated PCR products were immobilised in a microtiter plate coated with streptavidin (Combiplate 8, Labsystems, Helsinki, Finland) and the unbiotinylated strand was removed with alkali treatment ,15. The Primers and probes for the TaqMan assays were designed by Applied Biosystems as Assay-by-Design (rs1042719 ADRB2 and rs5925 LDLR) or Assay-on-Demand (rs1433099 LDLR) service. The probes for the two alleles were labelled with the reporter dyes FAM and VIC respectively. The sequences of the primers and probes for the SNPs rs5925 LDLR and rs 1042719 ADRB2 are found in . The priReal time quantitative PCR was run in 25 μl TaqMan Universal PCR Master Mix (Applied Biosystems) with 200 nM of both labelled TaqMan probes, 900 nM PCR-primers and 10 ng genomic DNA or one tenth of the cDNA products. The PCR conditions were initial activation of the enzyme at 95°C for 10 min followed by 60 cycles of 95°C for 15 sec and 60°C for 1 min in a ABI7000 instrument (Applied Biosystems, Foster City, CA, USA).The signal intensity ratios were calculated based on normalised ΔRn fluorescence values obtained from the assay during the exponential phase of PCR. The ΔRn values were retrieved from cycle 38 for the SNP rs1042719 ADRB2, cycle 42 for the SNP rs5925 LDLR and cycle 43 for the SNP rs1433099 LDLR. Imbalanced expression of the SNP alleles was determined by a t-test as described below.® Express instrument (Perkin Elmer Life Sciences, Boston, MA, USA) with the excitation lasers Blue Argon 488 nm, Green HeNe 543.8 nm, Yellow HeNe 594 nm and Red HeNe 632.8 nm with the laser power set to 80% and the photomultiplier tube gain adjusted to obtain equal signal intensities from reaction control spots for all four spectra. The fluorescence signals were extracted using the QuantArray ® analysis 3.1 software (Perkin Elmer Life Sciences, Boston, MA, USA). The mean of the fluorescence signals for the duplicate spots was corrected for the average background in each "sub-array" separately. The data was handled and interpreted using the Microsoft ® Excel program.In Methods I and II fluorescence was measured using a ScanArray 2) were assigned by linear regression analysis of the relationship between the signal intensity ratios determined from the minisequencing assay and the known allelic ratios in the mixed samples for the quantification standard curves. Two-sample t-tests with two-tailed significance levels assuming unequal variance were performed to determine the lowest level of detection of a specific allele for the quantification standard curves and to evaluate the imbalanced expression of the two alleles of the SNPs in the cell lines.The genotype for each individual SNP was assigned by calculating a ratio between the fluorescence signals for the two alleles. Coefficients of determination (RUL participated in the design of the study and in RNA and DNA extraction, and performed all the laboratory work involving "in-house" minisequencing methods, performed the statistical calculations and drafted the manuscript. MF cultured the cells, performed RNA and DNA extraction, performed the assays with the reference method, and provided input to the manuscript. AD performed the assays using the SNPstream system. A-CS conceived the study, participated in its design, coordination and in preparation of the manuscript. All authors read and approved the final manuscript.Additional file1 is a pdf-file with information on the SNPs, including dbSNP ID number, nucleotide variation and the sequences of the primers and probes used in the microarray based minisequencing and TaqMan assays respectively.Click here for file


The primate brain processes a remarkably diverse array of visual cues to recognize objects in dynamic settings crammed with unfamiliar objects. Not surprisingly, repeated viewing aids recognition, but how the brain orchestrates this experience-driven improvement is unclear. Visual input to the brain travels from the eye to the primary visual cortex (V1), at the back of the brain. From there, signals are sent to nearby extrastriate cortical areas, which process “early” visual cues. Both the “lower level” extrastriate cortex and “higher level” inferior temporal (IT) cortex are important for object recognition in primates. In monkeys and humans, lesions in the IT cortex severely affect the ability to recognize objects.In these higher-level cortical regions, neurons carry more information about an object after subjects learn to recognize that object. This modified neural activity is thought to reflect internal representations of specific aspects of the learned task—such as learned recognition of three-dimensional objects—and these representations often remain stable even though certain features of the visual stimulus—such as size or image degradation—change. With recent evidence suggesting that lower level brain regions like the primary visual cortex are also capable of learning-related modifications, it appears that both early and higher brain areas of the “ventral visual stream” benefit from learning. It is not clear, however, how learning modifies these discrete brain regions to coordinate this processing.By training monkeys to recognize degraded images, Gregor Rainer, Han Lee, and Nikos Logothetis of the Max Planck Institute for Biological Cybernetics in Germany have identified a subset of neurons that compensate for indistinct visual inputs by coordinating disparate regions in the brain. The monkeys' improved performance, they propose, stems from the informational enrichment of a subset of lower level neurons. Along with an increase in learning-induced firing activity, V4 neurons—extrastriate cortical neurons associated with detecting visual input of intermediate complexity—encode more information about relevant details to resolve indeterminate visual cues. V4 neurons likely interact with higher cortical levels to help the monkeys interpret the degraded indeterminate images as something recognizable.The researchers presented the monkeys with different “natural” images, including pictures of birds and humans, then subjected the images to different levels of “stimulus degradation”—making them harder to read by adding varying amounts of visual noise. Using this approach, the researchers could record the activity of the V4 neurons as the monkeys were presented with the different images. The monkeys viewed a sample image and then signaled whether a second image, presented after a brief delay, was a match or not. When Rainer et al. analyzed the activity of the V4 neurons associated with the different images, they found there was no significant change in the activity or information conveyed by V4 neurons associated with novel or undegraded familiar images. On the other hand, learning not only significantly improved the monkeys' ability to recognize degraded stimuli but also increased both the activity and informational encoding of the V4 neurons.But how did individual V4 neurons facilitate this enhanced ability to recognize degraded stimuli? After identifying a subset of neurons that showed enriched neural activity in response to degraded or indeterminate stimuli, the researchers studied the monkeys' eye movements to determine any behaviors that might explain why monkeys performed better with familiar degraded stimuli. They mapped the monkeys' eye movements while allowing them to freely view the different familiar and novel images—but this time with just two coherence levels (undegraded and 45% coherent). There was substantially more overlap, in terms of where the monkeys looked for the 45% and 100% coherent images after learning. This suggests that monkeys learned to focus their attention on particular salient features, and were thus better able to identify degraded versions of these images.Neurons in the V4 area appear to be recruited to distinguish the relevant visual signal from the visual noise, and thus play a critical role in resolving indeterminate stimuli when salient features are present. These results, together with previous studies showing the sensitivity of prefrontal cortex neurons to novel stimuli, indicate that the prefrontal cortex processes novel stimuli while the V4-rich extrastriate visual areas convey details about hard to decipher images. It may be that as the V4 neurons refine their competence through learning, they also support the ability of the prefrontal cortex to process different but similar visual cues. Vision is a dynamic process, Rainer et al. conclude, characterized by ongoing interactions between stimulus-driven brain regions and feedback from higher-order cognitive regions.


Tag-deleted SV40-derived vectors (rSV40s), since they do not elicit neutralizing antibody responses, and so can be given multiply without loss of transduction efficiency.A vaccine that elicits durable, powerful anti-HIV immunity remains an elusive goal. In these studies we tested whether multiple treatments with viral vector-delivered HIV envelope antigen (gp120), with and without IL-15, could help to approach that goal. For this purpose, we used recombinant SV(gp120) carried the coding sequences for HIV-1NL4-3 Env, and SV(mIL-15) carried the cDNA for mouse IL-15. Singly, and in combination, these two vectors were given monthly to BALB/cJ mice. Cytotoxic immunity and cytotoxic memory were tested in direct cytotoxicity assays using unselected effector cells. Antibody vs. gp120 was measured in a binding assay. In both cases, targets were P815 cells that were stably transfected with gp120.Multiple injections of SV(gp120) elicited powerful anti-gp120 cytolytic activity (>70% specific lysis) by unselected spleen cells. Cells from multiply-immunized mice that were rested 1 year after their last injections still showed >60% gp120-specific lysis. Anti-gp120 antibody was first detected after 2 monthly injections of SV(gp120) and remained elevated thereafter. Adding SV(mIL-15) to the immunization regimen dramatically accelerated the development of memory cytolytic responses, with ≥ 50% specific lysis seen 1 month after two treatments. IL-15 did not alter the development of antibody responses.Thus, rSV40s encoding antigens and immunostimulatory cytokines may be useful tools for priming and/or boosting immune responses against HIV. The development of an effective vaccine against HIV has been hindered by a variety of problems. The high mutation rate of the virus itself is such that it represents a moving antigenic target during the course of an infection -4. FurthCompared to administration of protein antigen or naked DNA, an infectious vector could be more effective at enhancing antibody and cytotoxic responses against a transgene product. Application of such a strategy, however, has been often complicated by the development of neutralizing immune responses, principally antibodies, against vector coat antigens -10. ThesTag-deleted SV40-derived gene delivery vectors (rSV40s) for immunization. Several studies have shown, both directly and indirectly, that rSV40 vectors do not elicit detectable neutralizing antibodies [in vivo.This limitation can be circumvented by repeatedly changing the serotype of the antigen-carrying vector, or by using recombinant tibodies -13. Eventibodies ,12 or diTag-deleted rSV40 vectors (unlike wild type SV40), capsid proteins are not expressed, immune responses can only be generated by transgene products [The explanation for this unusual state of affairs may lie in the fact that SV40 enters cells via caveolae and thence travels directly to the nucleus, bypassing cellular antigen processing -16. Thusproducts ,12. Whetproducts ,14. We hOther investigators have reported that co-administration of vectors carrying immunostimulatory cytokines was useful in augmenting anti-lentiviral immune responses -19. IL-1Both antibody and cell-mediated immune responses may be useful to protect from HIV infection and progression to AIDS -26. HoweWe used rSV40s to study the generation and longevity of both humoral and cell-mediated responses in an effort to generate immune responses against the HIV-1 envelope glycoprotein, gp120. We also tested whether co-immunization regimens involving rSV40 delivery of both IL-15 and gp120 augmented and/or accelerated SV40-mediated immune responses further.The murine mastocytoma cell line P815 (ATCC, Bethesda, MD, USA) was used, and maintained in culture with Dulbecco's Modified Eagle's medium (DMEM), supplemented with 10% newborn calf serum (NCS) (Gibco BRL/Life Technologies, Grand Island, NY, USA). COS-7 cells (ATCC, Bethesda, MD, USA), were used to expand stocks of recombinant SV(gp120) and SV(mIL-15) viruses. Cytotoxic lymphocytes were obtained from spleens of immunized mice, and cultured in RPMI-1640 (Gibco BRL/Life Technologies, Grand Island, NY, USA) supplemented with 10% NCS (RPMI-10). Rabbit kidney fibroblasts (RK13 cells) and CV-1 cells (African green monkey kidney cells) were obtained from ATCC (Bethesda, MD, USA). RK13 cells were used to propagate stocks of VCB41, a vaccinia virus vector carrying HIV-1NL4-3 envelope gp120 sequence.BALB/cJ mice aged 6–8 weeks were purchased from Jackson Laboratories, Bar Harbor, ME, USA. They were fed and housed in accordance with American Association for Accreditation of Laboratory Animal Care standards. Use of mice in the laboratory protocols described was approved by the Thomas Jefferson University Institutional Animal Care and Use Committee.A 1.6 kb DNA fragment encoding gp120 from HIV-1NL4-3 was made by PCR using primers with engineered restriction sites. This PCR product was cloned into pT7A5  immediate early promoter and downstream polylinker), giving pT7A5-gp120. To make SV(gp120), the SV(gp120) genome was released from the carrier plasmid by restriction digestion, and used to make recombinant virus in COS-7 cells as described previously . Virus sTag-deleted SV40 genome [[et al., in preparation], to yield prSVmIL-15. Virus was made from this plasmid in COS-7 cells as previously reported [To generate a recombinant SV40 virus with the murine IL-15 transgene (SV(mIL-15)), mIL-15 cDNA was cloned into pSL-4p, which contains a  genome [, Vera, ereported .9 infectious units (IU) of SV(gp120) ± SV(mIL-15) intraperitoneally (IP). In some studies, final administrations included both IP and subcutaneous (SQ) inoculations. SV(HBS) was used as a control antigen-carrying vector. Specific immunization schedules are described in the Results section, below.Mice were given monthly 1 × 10Production of HIV Env-expressing stably transfected targets is similar to the procedure used for generating SIV Env-expressing targets . BrieflyFlow cytometry was used to verify gp120 expression on the surface of P815 cells. A recombinant vaccinia virus carrying HIV-1NL4-3 gp120 (VCB41, NIH AIDS Reference Reagent Repository Program (NIH-ARRRP)) was used both as a positive control for gp120 expression and also to generate gp120-expressing target cells in some experiments. Cells that had been stably-transfected with plasmid gp120, or infected with VCB41 both expressed gp120, as assayed by flow cytometry (Coulter-Epic, Kimmel Cancer Center, TJU) (data not shown).The gp120-expressing P815 population was then cloned by limiting dilution. Clonal outgrowths were then reanalyzed by cytofluorimetry (FACS, data not shown) and the single clone expressing the highest levels of gp120, clone 24, was used in subsequent studies as a target for cytotoxicity assays.405nm of test sera vs. VCB41-infected P815 cells, subtracting A405nm due to binding to wild type (wt) VV-infected P815 cells, and also subtracting A405nm of control sera from mice immunized in parallel with a control rSV40, SV(HBS)An ELISA method was used to assay the activity of anti-gp120 antibodies elicited by immunization of the mice with SV(gp120) ± SV(mIL-15). The strategy for our appraoch to testing for antibodies vs. HIV Env is similar to one we have used to measure binding activity vs. SIV Env [Wild type (wt) P815 cells, or clone 24 P815 cells expressing gp120 were the target cells for unselected lymphocytes from spleens of mice immunized with SV(gp120) (± SV(mIL-15)), or SV(HBS) as control. Where SV(gp120) was used alone, P815 cells infected with wt vaccinia virus or VCB41 were target cells for spleen cells of immunized mice.9 IU intraperitoneally (IP) and 1 × 108 IU subcutaneously (hind footpads) usually 4 d before assay, but up to 1 month prior to assay, to test cytolytic lymphocyte memory. Spleen cell concentrations adjusted to 2 × 106 / ml with RPMI-10.Mice were boosted simultaneously with 1 × 10In some assays of cytotoxic lymphocyte memory, effector cells were harvested 1 month after the final injection. Effector cells from immunized mice were prepared as described, but in addition, were incubated with 5 μg/ml Concanavalin A  overnight, prior to assay. Con A-stimulated cells were then harvested, washed once in RPMI-10, and then used with target cells in the assay.51Cr  (100 μCi per 1 × 106 cells) at 37°C, 5% CO2 for 4 h as described previously [2 for 4 hours. Supernatant 51Cr was counted (1282 Compugamma CS, LKB) [P815 target cells were washed, then labeled with eviously . Afterwa51Cr from wild type target cells was subtracted. Thus:Mean c.p.m. for gp120-immunized effector cells mixed with gp120-expressing targets, minus the mean c.p.m. control (SV(HBS))-immunized effector cells vs gp120-expressing targets, and expressed as a percentage of the maximal target cell lysis (target cells incubated with 1% Triton-X). Background release of 51Cr release = {[c.p.m. 51Cr released by SV(gp120)-immune populations from gp120-expressing P815 cells] minus [c.p.m. 51Cr released by SV(HBS)-immune lymphocytes from gp120-expressing P815 cells]}, divided by [c.p.m. 51Cr released by Triton X-100 from gp120-expressing P815 cells]. The same calculations were done for lysis of wild type P815 cells by gp120-immune and control-immune effector populations. These numbers were then subtracted from the calculated 51Cr release above to determine the gp120-specific lysis of target cells by SV(gp120)-immunized effector cells.% specific P815 cells were transduced with SV(mIL-15) ×1 at m.o.i. = 100. Culture supernatants were harvested at several times post-transduction, and stored at -80°C. At day 6 post-transduction, a well of cells was harvested and lysed (2% NP40, 50 mM Tris pH7.4,150 mM NaCl, 1 mM EDTA, 10% Glycerol + protease inhibitor cocktail (25× stock Complete™ EDTA-free protease inhibitor cocktail, Roche Diagnostics GmbH, Mannhein, Germany)). Remaining wells were activated non-specifically with 5 mg/ml Con A, and supernatants harvested at various times thereafter. 3 days after con A stimulation, cells were lysed as described above. 50 μg of each culture supernatant or lysate were loaded on a 4–20% Tris-HCl gradient gels (Ready Gel, Bio-Rad, Hercules, CA, USA). 50 ng recombinant human IL-15 was used as a positive control. Samples were electrophoresed, and blotted to PVDF membranes (Immobilon™-P, Millipore Corporation, Bedford, MA). Blots were blocked overnight at 4°C with 5% milk in PBS + Tween-20 (0.05%). Rabbit anti-mouse IL-15 (Abcam, Cambridge, UK) was used as primary antibody, (diluted 1:500 with PBS-Tween), for 2 h at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) was used at 1:10,000 dilution in PBS-Tween, for 1 h at room temperature. Signal was detected with chemiluminescence reagent COS-1 cells were infected with SV(mIL15) or SVLUC (carrying luciferase) as a negative control. 24 h later, the media were changed and cells incubated 48 h in 500 μl of RPMI 10% serum/well. Fresh mouse spleen cells (5000/well) then cultured 48 h with 100 μl of cell supernatant + 100 μl of RPMI 10% serum. IFNγ ELISA (Pharmingen) was performed on the supernatant from these cultures.Methods). We used flow cytometry to identify the clone most strongly positive for cell membrane gp120. Compared to other stably-transfected clones, "clone 24" expressed gp120 at the cell membrane best (data not shown). VCB41-infected and SV(gp120)-transduced P815 cells also expressed substantial cell membrane gp120. Control wtP815 cells, or P815 cells infected with wt VV did not (data not shown).P815 cells stably transfected to express HIVNL4-3 gp120 were selected and cloned by limiting dilution )-immunized rSV40-immunized mice vs. clone 24 cells. Thus, data presented below reflect gp120-specific responses against clone 24.Methods. Specific binding antibody activity was first statistically significant, compared to prebleed sera 2 weeks after the second inoculation of SV(gp120) (P = 0.000332, using two-tailed Student's t-test) and reached a plateau after the third inoculation  , and their sera were assayed for reactivity vs. gp120 by CELISA. The details of this cell-based ELISA, or CELISA, as described in 20, and tAn effective anti-lentiviral immunization regimen should generate cytotoxic memory cells. To see if SVgp120) treatment could do this, mice were immunized once with SV(gp120) IP, then sacrificed 1 month later, without further treatment. In order to lyse target cells, committed cytotoxic cells require activation. However, to avoid antigen-specific selection and specific stimulation only of gp120-reactive cytotoxic cells, splenic lymphocytes were non-specifically stimulated by overnight incubation with Con A. A single immunization with SV(gp120) alone elicited only weak memory lytic responses (≤ 10% specific lysis) against gp120-expressing target cells  one month after the first, then were assayed the same way one month later for anti-gp120 cytolytic activity. These mice made stronger specific memory responses (15–20% specific lysis) than did animals given only a single inoculation (P ≤ 0.04, by Student's t-test, comparing 2 injections with just one)  gp120-specific cytolytic responses (mean specific lysis of 61% ± 4.2).To test whether SV(gp120) could elicit very long term cytotoxic lymphocyte memory, mice were immunized monthly ×8 with SV(gp120) IP. A final IP inoculation with SV(gp120) was given Methods, using rabbit antibody vs. murine IL-15 , and lower levels were seen with 2 injections, we tried to accelerate development of such responses using IL-15, delivered by transduction. To determine if IL-15 could be expressed by transduction, P815 cells were transduced with SV(mIL-15) at m.o.i. = 100. Culture supernatants were harvested 36, 72 and 144 hours later, at which point the cultured cells were activated with Con A. Culture supernatants were collected at 24 and 72 hours post-activation. Supernatants were assayed for IL-15 secretion by Western analysis as described in The functionality of the IL-15 produced in this fashion was tested by exploiting the ability of IL-15 to elicit production of IFN-γ by lymphocytes. Thus, CV-1 and COS-7 cells (African green monkey kidney cells) were transduced by SV(mIL-15), then cultured for 72 hrs. Control cultures of the same cells were transduced with SVLUC (carrying luciferase as a transgene). Normal mouse spleen cells were cultured for 42 hrs in 200 μl of the resulting culture supernatants. Production of IFN-γ by the spleen cells was measured by ELISA. Supernatants from COS-7 and CV-1 cells elicited respectively 2056 ± 363 pg/ml and 880 ± 196 pg/ml IFN-γ. Supernatants from SVLUC-transduced cells did not elicit detectable interferon secretion by spleen cells (<20 pg/ml).9 IU of rSV40: one group was given SV(mIL-15) alone, and one group SV(gp120) alone. Three other groups received both SV(mIL-15) and SV(gp120): either SV(mIL-15) followed 3 days later by SV(gp120), or SV(gp120) first, followed by SV(mIL-15). The final group was given both SV(mIL-15) and SV(gp120) simultaneously. The 3 day separation between the two vectors was used because of the strength of the signal for secreted IL-15 by Western blotting at 72 hours post-stimulation (see above). One month later, unselected spleen cells were assayed as described above for cytolytic activity against gp120-expressing clone 24 cells.To determine whether coordinate administration of SV(mIL-15) plus SV(gp120) improved cytolytic responses against gp120, mice were given two sets of injections IP, one month apart. Normal BALB/c mice received IP with 10Adding IL-15 to the immunization regimen greatly increased gp120-specific cytolytic responses Figure . Also, tMethods.Mice receiving SV(mIL-15) and/or SV(gp120) (or the control vector, SV(HBS)) according to the schedules outlined above were tested to determine the effect, if any, of such co-administration in anti-gp120 serum antibody responses. CELISA and calculation of gp120-specific antibody binding were performed as described in Slight binding antibody activity was detected 2 weeks after the first inoculation(s), in all SV(gp120) recipient groups. Levels of SV(gp120)-induced groups made detectable antibody responses were not appreciably affected by coadministration of SV(mIL-15) (data not shown).in vivo. Our results here demonstrate several important strengths of using rSV40 vectors to immunize against lentiviral antigens: Among these are the ability of the vector to be administered multiple times without eliciting neutralizing responses [51Cr-release assays: unselected lymphoid organ populations were added directly to labeled target cells at low E:T ratios, and specific 51Cr release was measured.In this study, we used rSV40 vectors to elicit HIV-1NL4-3 gp120-specific cytotoxic lymphocyte and antibody responses. We have observed that these vectors may be administered repeatedly to boost those responses. Further studies also suggested that such responses are durable esponses -13, and 51Cr release observed in the current studies were extensively controlled to ascertain the antigen-specificity of the cytolysis observed: background lysis of wild type P815 cells was subtracted, as was lysis by lymphocytes from mice immunized with an irrelevant rSV40 vector. We also found that cytolysis increased with increasing numbers of immunizations, which is not a characteristic of NK cell-mediated lysis.Analysis to confirm CD8 expression, or expression of other CTL markers was not performed on the effector cells. However, it is unlikely that these data reflect the cytotoxic activity of NK cells. NK cytolytic activity is non-specific and does not increase with repeated immunization. The patterns of in vivo, we tested whether cytotoxic lymphocyte activity elicited by SV(gp120) immunization, was detectable one month after inoculation. Thus, cytolytic responses, assayed one month after a second injection, were ≈ 20%, which is comparable to those of splenic cytotoxic cells assayed four days following a third inoculation (data not shown). Further, mice given multiple injections of SV(gp120), then rested for one year, gave ≈ 70% specific lysis when challenged with SV(gp120). Therefore, SV(gp120) administration may thus favor development of cytotoxic lymphocyte memory.Since a key goal for a vaccine against HIV is to generate immune responses that are durable In an attempt to accelerate and to improve upon these specific cytotoxic and particularly cytotoxic memory responses, we co-immunized mice with SV(gp120) and a rSV40 carrying mouse IL-15. IL-15 promotes cytotoxic lymphocyte responses, and in particular, cytotoxic memory responses ,24. The in vitro. Furthermore, low effector:target ratios (20:1 and 10:1) were used in these assays. Our immunization protocols tested both simultaneous and staggered administration of rSV40s carrying HIV-1NL4-3 gp120 and murine IL-15.Accordingly, our analysis of the contribution by IL-15 to cytotoxic responses, focused mainly on the ability of SV(mIL-15) to augment specific cytotoxic responses of spleen cells from animals rested 1 month following immunization. Because quiescent cytotoxic T cells are not strong effectors, we non-specifically activated the splenocytes prior to assay with Con A. Non-specific activation was used to avoid specifically enriching effector cell population for gp120-specific cells IL-15 co-immunization dramatically accelerated cytotoxic responses, depending on the immunization regimen used: Animals given SV(mIL-15) alone made no gp120-specific cytolytic responses. Mice receiving 2 treatments with a mixture of SV(gp120) and SV(mIL-15) gave much higher specific lysis, depending on the coadministration regimen, as compared to those receiving SV(gp120) alone (≈ 10% specific lysis). Thus, among mice given staggered injections of SV(mIL-15) and SV(gp120), the order of cytokine administration greatly affected the response: if SV(gp120)was given first, no detectable gp120-specific cytolysis was observed. However, if the cytokine was given first, followed 3 days later by SV(gp120), ≥ 60% specific lysis was seen at both 20:1 and 10:1 effector:target ratios.Why the order of cytokine administration should affect antigen-specific responses so dramatically is not yet clear. Cytokine given after, or together with antigen, may have insufficient time to augment cytotoxic responses. In addition, Western analysis of IL-15 production showed that IL-15 secretion was not detectable in supernatants beyond 36 hours, but could be stimulated subsequently. Thus, a specific, possibly brief, window for IL-15 expression and secretion may need to be attained, in order for its effects on gp120-specific responses to be detectable. We observed very high levels of specific lysis by these unselected effector populations following just two tandem injections of SV(mIL-15) followed by SV(gp120).The strong anti-lentiviral cytolytic responses we report were observed in a strain of mouse, BALB/cJ, that generally mounts relatively weak type 1 T cell responses. The finding of >60% cytolysis with two administrations of SV(gp120) + SV(mIL-15), suggests that a strategy similar to that described herein may be helpful in individuals who would generate relatively low cytolytic responses.Serum antibody levels assayed by CELISA where SV(gp120) was administered alone, multiple times, were detectable after two immunizations, and continued to increase up to week 4 following the third immunization. These responses were not further enhanced by subsequent boosting immunizations. While specific antibody responses against gp120 were detected in all experimental groups following SV(gp120) and SV(mIL-15) co-immunization, IL-15 co-administration did not augment anti-gp120 antibody levels, compared to gp120 alone. This was to be expected, since IL-15 reportedly acts primarily on T cell and NK cell functions, rather than on humoral immune responses.Our data argue in favor of using IL-15 as an adjuvant for antigen-specific immune responses, particularly cytotoxic lymphocyte responses. We also demonstrate that a single transgene, administered multiple times (>3), may be very effective at eliciting both humoral and cell-mediated responses. These results thus both corroborate and extend our previous observations ,14,32, aRecombinant SV40-derived gene-delivery vectors, being transparent to the immune system, can be given multiple times to prime and boost immune responses against the delivered antigens. Anti-vector immunity does not overwhelm responses against the target antigens. As well, these vectors elicit very high levels of antibody, and especially cell-meditated immunity. Finally, combining the delivery of rSV40s bearing antigens with those bearing cytokines such as IL-15 can enhance levels of immunity, particularly long-term immunity. Clearly, much work remains. However, this approach offers promise as a strategy to immunize against pathogens for which classical approaches have not been adequately effective.None declared.HJM devised all the assay systems for cell- and antibody-mediated immunity against lentiviral antigens, performed all the immunization studies and assays. HM also wrote this manuscript. PYT generated the SV(gp120) construct. MV and PF generated the SV(mIL-15) and SVLUC constructs described here and performed the ELISA for IFNγ stimulated by SV(mIL-15). DSS is the Principal Investigator for this work, oversaw and planned the experimental strategies, worked with HJM in interpreting the experimental data and writing the manuscript.


Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4–6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.Integration of mitochondrial DNA fragments into nuclear chromosomes  is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of  DNA from mitochondria has regularly inserted into the human nuclear genome. Some insertions are polymorphic, revealing that the invasion of the human genome is an ongoing process Insertion of new sequences into nuclear DNA has a major impact on its architecture and is an important mechanism for the evolution of eukaryotic genomes. Moreover, when targeted to gene loci, these insertions can be mutagenic, and in humans this process contributes to a number of diseases . The freDNA fragments of mitochondrial origin, originating from both coding and noncoding regions, are found as sequence fossils in the nuclear genomes of various eukaryotes . HoweverHomo sapiens, experimental approaches that compare individuals within this species and its closest relative, the chimpanzee, can be undertaken .Our updated analysis (data not shown) reveals that the majority of these NUMTs correspond to those previously documented . HoweverTo determine whether sequences of mitochondrial origin were actually integrated in the human nuclear genome, and were not a result of contamination of DNA library preparations, we selected 42 NUMTs for analysis in human samples. Our choice included the 36 NUMTs with the highest identity (91% to 100%) to the mtDNA, one NUMT having the longest stretch of DNA with high identity (88%), one NUMT corresponding to the highly variable region of the mtDNA (D-loop) , and fouIn summary, results from two amplification strategies and from sequencing demonstrated that these NUMTs were indeed present at the expected chromosomal location and that they are bona fide mt sequences residing in the human nuclear genome.The colonization of human populations by various NUMTs revealed striking disparities. Thirty-five NUMTs were present in homozygous form in all individuals tested see . InteresInterestingly, one or more of these six NUMTs were detected among individuals within each ethnic group, indicating that their insertion in the nuclear genome occurred soon after the origin of modern humans and that they represent the most recent integrations of our studied cases. Despite the limited sampling size , the frequency of alleles carrying the insertion varies greatly according to the NUMT . For each locus, one to three chimpanzee individuals were analyzed. Forty-two out of 42 primer pairs successfully amplified the target site also in chimpanzees because of the high sequence identity of the two genomes (average 98.7%)  are unlikely to be human-specific. This results in an average of one NUMT integration in the germline for each 180,000 y, in the last 4–6 million years (Myr). Interestingly, one fourth of these NUMTs (6/27, or 7/28 if we include NUMT 11-541) show insertion polymorphism  . Only the 98.7%) . All of The distribution of human-specific NUMTs in human chromosomes is not proportional either to the chromosome size or to the total number of NUMTs present in the chromosome . In ChroQ8N7L5 into two, and the NUMT itself becomes a new intron , a species closely related to humans and whose evolutionary relationship with humans has been widely investigated, is an ideal candidate for a comparative analysis. Moreover, the high level of identity (more than 98%) between the two species (H. sapiens. For NUMTs fixed in the human genome (not displaying insertion polymorphism), we do not expect this value to increase significantly, since our analysis was made on essentially the entire human genome.An important question concerning the integration of DNA sequences in the nuclear genome is their rate of colonization. For exogenous sequences like NUMTs, this has not been investigated in vivo. To determine the extent of colonization of a given genome, it is necessary to compare the insertions within this genome with those of a closely related species. To date, a comprehensive analysis of the presence of NUMTs has been done for several complete nuclear genomes , but the species  allows tH. sapiens and have integrated in the human genome in the last 4–6 Myr, after the split of the two species from their common ancestor (Homo erectus out of Africa (1.7 Myr ago). Living humans would still be polymorphic for these NUMTs, as a result of interbreeding of the nonmodern human populations with modern humans  are specific to ancestor . This coancestor . Howevern humans . On the n humans . In thisCompared to 28 NUMT insertions in the human nuclear genome in the last 4–6 Myr, it has been calculated that about 5,000 new insertion events of Alu repeats have occurred in the human genome in the same timescale  “BLAST the Human Genome” server (http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs). The blastn program was used with default parameters on April 24, 2003. Only output parameters were changed to 1,000 descriptive lines and to 1,000 segment alignments. In a few cases  were obtained as purified DNA from Coriell Institute (Camden, New Jersey, United States). Purified chimpanzee DNA, obtained either from tissues or from fecal material, was a kind gift from J.-P. Vartanian at the Pasteur Institute (Paris, France). For both PCR strategies described in the text, primer sequences are available upon request. Cell lysis was performed by incubating fresh cells overnight at 55 °C in a Tris-EDTA buffer (pH 8.5) in the presence of 200 μg/ml of proteinase K. PCR amplification was performed with 30 cycles of denaturation (1′ at 94 °C), annealing (1′ at 68 °C), and DNA synthesis (3′ at 72 °C) using Invitrogen  −8 mutations per nucleotide per generation, or 1.25 × 10−9 mutations per nucleotide per year, assuming a generation time of 20 y (−8 substitutions per nucleotide per year, excluding the D-loop (i, is given by i = d/(μM + μN), where d is the frequency of sequence divergence between the NUMT and present mtDNA sequence. As an example, for a sequence 300 bp long, 94% identity to mtDNA corresponds approximately to an insertion time of 3.3 Myr, and 96% to 2.2 Myr.The age of insertion of NUMTs was estimated using, as reference, the sequence divergence of the NUMT from the mtDNA. We assumed that the NUMT, when inserted into the nuclear genome was identical to the corresponding mt sequence. We also assumed that, once inserted into the nuclear genome, the NUMT mutated at the same rate as the nuclear genome, μN, which corresponds, for noncoding sequences, to 2.5 × 10 of 20 y . By compe D-loop . Thus, fTable S1(53 KB DOC).Click here for additional data file.Table S2(63 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs) accession number for the human mtDNA sequence is AB055387.The NCBI (