Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants.An cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case.A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both  Escherichia coli strain in which RecBCD has been genetically replaced by Red exhibits greatly elevated levels of recombination between its chromosome and short linear double-stranded DNA species sharing sequences with the chromosome [The Red recombination system of bacteriophage λ promotes efficient double strand break repair/recombination. An romosome .cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant [red and the bacterial recombination genes recA, recF, recO, recR, recQ, ruvAB, and ruvC. Large numbers of chloramphenicol-resistant Lac+ recombinants were generated in these crosses as well. In this study, we characterize the Lac+ recombinants and the processes which generate them. Most appear to arise from events in which homologous recombination between the incoming DNA and the chromosome generates a partial duplication of the bacterial chromosome.In previous studies, we have characterized a recombination event, pictured in Figure ombinant -4. The drecA, recF, recO, recR, recQ, ruvAB, and ruvC. Deletion of recG increased the production of both Lac+ and Lac- recombinants. Lac+ recombinants accounted for 10–60% of the total chloramphenicol-resistant offspring of the crosses. These results strongly suggest that formation of the Lac+ recombinants takes place via homologous recombination, though they do not rule out the possibility that non-homologous end joining, or other varieties of "illegitimate" recombination, might be involved as well.Data presented in Table lac::cat sequences would be attached to phage sequences. Heitman et al. [in vivo are rapidly repaired by DNA ligase. The results of previous physical studies with other substituted λ phages cut in vivo by PaeR7 lead to the expectation that the λ lac::cat819 chromosome would be uncut, or only singly cut, much of the time in the infected cell [The reason for considering mechanisms other than homologous recombination in the formation of the Lac+ recombinants is because of the expectation that some of the phage-borne n et al.  showed tted cell ,6.lacZ (not shown). When the lac::cat dsDNA was delivered into the cell by a λ vector bearing a tetracycline resistance determinant (Δ nin::tet859 – described below) neither the Lac+ nor Lac- recombinants acquired tetracycline resistance. These findings suggest that the process which formed the recombinants did not involve the entire phage chromosome.Preliminary characterization of the Lac+ recombinants formed in cells which were wild-type for all the recombination functions revealed that most, possibly all, were unstable. When streaked on plates containing chloramphenicol and X-Gal, they segregated Lac- (colorless) progeny at variable frequencies. Southern gel analysis of chromosomal DNA from unstable Lac+ recombinants revealed that some of them were multiploid for lac locus, will produce a Lac+, chloramphenicol-resistant recombinant. Data presented below, in which simpler lac::cat recombining substrates generate Lac+ recombinants at a frequency approximately 1000-fold lower than Lac-, suggest the frequency of spontaneous lac duplications in Red+ but otherwise wild type E. coli is approximately 10-3, consistent with estimates of spontaneous duplication frequency in Salmonella made by Roth et al. [A baseline level of Lac+ recombinants was to be expected in these crosses. The recombination event pictured in Figure h et al. . The Laclac::cat segment were to recombine with more than one chromosome at a time. Further complicating interpretation of the experiment, the cells were infected at a multiplicity of 10 phages per cell.The crosses described above were done by infecting log phase host cells grown in rich medium. Such cells contain multiple copies of their chromosome. Complications might arise if the lac::cat segment and single copies of the bacterial chromosome. As shown in Table To reduce the complexity of the system, we switched to a cross procedure involving low multiplicity infection (0.1 phage per cell) of stationary phase cells. Presumably, under these conditions, most of the events producing chloramphenicol-resistant recombinants take place between single copies of the lac::cat, which are diagrammed in Figure lac::cat819, the progenitor of this series, has two PaeR7 sites. Upon infecting a host cell bearing the Δ recBCD::Ptac-gam-bet-exo-pae-cI substitution, it injects its chromosome, which circularizes. Expression of its lytic genes, including those necessary for phage DNA replication, is blocked by the action of cI repressor. Cutting of its chromosome by the PaeR7 restriction endonuclease in the infected cell releases a 3.5 kbp linear dsDNA, consisting of the cat gene and 1.3 kbp flanks of lac sequences on the right and left. . The ends of the flanking lac sequences match precisely their counterparts in the chromosome.To examine the dependence of Lac- and Lac+ recombinant formation on the structure of the DNA substrate, we constructed variants of λ lac::cat930 and λ lac::cat931 have only single PaeR7 sites, on the right and left, respectively. Cutting by PaeR7 in the infected cell should produce large linear dsDNAs related to the lac::cat819 fragment, but with long tails of non-homologous DNA extending from the left and right sides, respectively. Interestingly, both of these single-cut phages produce recombinants more efficiently than the double-cut λ lac::cat819, in both recG+ and Δ recG backgrounds  Some of the λ lac::cat929 chromosomes might be cut by the EcoK restriction endonuclease. The infected cells contained a functional EcoK restriction-modification system. λ lac::cat929 was grown in a similarly EcoK+ host, and therefore was presumably EcoK-modified. However, if the modification were not complete, there might be residual EcoK restriction activity, resulting in 10% of the phage chromosomes being cut. . (2) The production of the recombinants might not be Red-mediated. (3) The linear partner might be a broken bacterial chromosome.Approximately half of the strongly chloramphenicol-resistant recombinants generated by the uncuttable phage λ  partner . How, thhsdR, with a tetracycline resistance determinant. This replacement had little effect on the formation of recombinants by λ lac::cat929 . A fourth event, also not shown, involves recombination between λ genes and homologues in cryptic prophages in the E. coli chromosome (discussed below). As suggested in the figure, cointegrates formed by recombination in the right-side lac flank are phenotypically Lac-, while recombination in the left-side lac flank (or other loci) forms Lac+ recombinants. To test this idea, we constructed variants of λ lac::cat missing either or both lac flanks; they are diagrammed in Figure Cointegrates could in principle be formed by four different single reciprocal recombination events between the circular λ cat cassette with 1000 bp homologous flanks was only 10-fold more efficient than one with 40 bp flanks. Their experiments were done with cells which had been subjected to heat shock and electroporation. To test the efficiency of short-flank recombination under less extreme conditions, we constructed a short-flank λ lac::cat variant.The ability of the λ Red system to promote recombination events involving short sequence homologies makes it particularly useful for genetic engineering. Yu et al.  reportedlac::cat921 consists of the cat gene flanked by 40 bp sequences corresponding exactly to the terminal 40 bp at each end of the 1.3 kbp flanks of λ lac::cat819. As shown in Table lac::cat921 exhibits a several hundred-fold lower efficiency of recombinant formation than its long-flank counterpart, in both recG+ and Δ recG backgrounds. λ lac::cat921 was similarly inefficient in log phase cells (data not shown). These observations suggest that short-flank recombination may not be a significant activity of the Red system in nature. However, they do not speak to the question of whether long flanks work better because they provide a larger homology target for synapsis, or because they provide non sequence-specific protection, perhaps delaying exonucleolytic degradation of the recombining sequences long enough to permit recombination to take place.The fragment released by PaeR7 from the chromosome of λ lac::cat930, but not by the left side-cut λ lac::cat931  cloning the cat gene from the deletion mutant, along with flanking chromosomal sequences; (3) using the cloned, plasmid-borne flanking sequences, and Red-mediated gap repair, to clone the lac operon in a plasmid; (4) inverting the lac operon, relative to its flanking sequences, in the plasmid; (5) replacing the Δ lac::cat chromosomal allele with the plasmid-borne lac-inv allele.The strategy for inverting the chromosomal lac::cat930 and lac::cat931 alleles were constructed by replacing the left-side and right-side PaeR7 sites, respectively, of pTP819, with XbaI sites. To produce inverted derivatives of these two alleles, the plasmids pTP930 and pTP931 were digested with XbaI and XhoI (a PaeR7 isoschizomer); lac::cat inserts and bla-ori backbone fragments from the two plasmids were exchanged. The inverted alleles were then crossed into phage λ, as described in the Methods section.The single-cut E. coli K-12 [lac::cat930 and λ lac::cat931, and the efficiency of Lac+ recombinant formation by λ lac::cat930, were slightly elevated relative to those in the corresponding AB1157-derived strain.In constructing the chromosomal inversion, we switched genetic backgrounds, from the AB1157-derived strains with which most recombination studies have been done, to MG1655, the sequenced wild type oli K-12 . In the lac-inverted E. coli) were tested for Lac+ recombinant formation. The results are shown in Figure lac::cat930  in lac-wild type, and lac::cat1033  in lac-inv.The eight combinations of normal and inverted phages and bacteria  lacking its sequences derived from phage P22 was equally proficient at generating Lac+ recombinants. A derivative missing the left lac flank made Lac+ recombinants almost exclusively; the small number of Lac- recombinants in this case probably represent cointegrates made by uncut plasmid DNA in the fragment preparation.We constructed several other linear DNA substrates to test the generality of the model in Figure ion  and λ lac::cat930 (cut right) behave like their counterparts generated by electroporation of linear dsDNAs into Red+ cells: they segregate Lac- chloramphenicol-resistant clones at low frequency, and Lac+ chloramphenicol-sensitives at high frequency (data not shown). In addition, their formation also depends upon the presence of cryptic prophages in the chromosome: λ lac::cat819 was found to produce Lac+ chloramphenicol-resistant recombinants as only 0.13% (average of six measurements) of the total chloramphenicol-resistant progeny in crosses with TP750. These properties suggested both kinds of recombinant might have the same structures as well, but PCR tests of 50 of the phage-generated recombinants with the primers used to demonstrate type I, II, and III recombinants  when electroporated directly into cells .One aspect of Lac+ recombinant formation by λ ls Table . How doelac::cat-bearing λ phages and the bacterial chromosome, as well as the mechanism of their formation. Our studies uncovered a diversity of recombinant structures, including complex duplications and cointegrates. Recombination between λ and cryptic prophage sequences in the bacterial chromosome was found to be the most significant mechanism generating Lac+ recombinants. All of the Lac+ recombinants could be generated by homologous recombination events of types which have been previously described. In particular, there was no evidence for end-joining or other non-homologous recombination events. Perhaps the most surprising finding was the high frequency with which large duplications in the bacterial chromosome – up to 1 Mbp – could be generated by the Red system.The bacteriophage λ Red recombination system is of general interest for two main reasons. First, it is an intensively characterized and relatively simple system, which serves as a model for studies of homologous recombination -17. SecoE. coli strain DH5α (λ pir) [hsdR::tet allele in TP842 was constructed by using a Tn 10-containing E. coli strain as template in PCR with primers hsdRut (5'-TTGGACAGGCCCGCACAGCAATGGATTAATAACAATGATGCTCGACATCTTGGTTACCGT-3') and hsdRdt (5'-GCTGAATTTGCCCAGCAGGGTATCGAGATTATCGTCAAAGCGCGGAATAACATCATTTGG-3'). The Δ lac::cat allele in TP872 was constructed by using a Tn 9-containing E. coli strain as template in PCR with primers cat15 (5'-TCTGGTGGCCGGAAGGCGAAGCGGCATGCATTTACGTTGAATGAGACGTTGATCGGCACG-3') and cat16 (5'-AGAGTACATCTCGCCGTTTTTTCTCAATTCATGGTGTACAATTCAGGCGTAGCACCAGGC-3'). (λ pir)  was usedcI857 Sam7 containing the nin region genes was cloned into the EcoR1 site of pBR322. In the resulting plasmid, pnin, the nin genes are read clockwise in the conventional map of pBR322. pTP772 was constructed by deleting sequences between the two HindIII sites of pnin. pTP859 was constructed by cutting pTP772 with SacII and ClaI, blunting the ends with T4 DNA polymerase, ligating with NotI linkers (5'-AGCGGCCGCT-3'), cutting with NotI, and ligating with a NotI fragment of pTP857 [tetR and tetA genes of transposon Tn10.An EcoR1 fragment of λ f pTP857  containicat gene is flanked on both sides by lac sequences, PaeR7 sites, and λ sequences (for crossing into the phage), has been described [lac flank and PaeR7 site. pTP922 was constructed by deleting the lac and cat sequences between the two PaeR7 sites in pTP819. . pTP926, pTP927, and pTP928 were constructed by ligating the oligonucleotide 5'-TCGACAGTCTAGACTG-3' into the PaeR7 sites of pTP828, pTP829, and pTP922, respectively, eliminating the PaeR7 sites and replacing them with XbaI sites. pTP929 was constructed by ligating the XbaI-NcoI fragment of pTP926 containing the N-terminal coding sequences of cat and the NcoI-XbaI fragment of pTP927 containing the C-terminal coding sequence of cat into the XbaI site of pTP928. The orientation of the reconstructed cat gene is the same as in pTP819. pTP930 was constructed by ligating together the large ApaI-SacII fragment of pTP929 and the small ApaI-SacII fragment of pTP819. pTP931 was constructed by ligating together the small ApaI-SacII fragment of pTP929 and the large ApaI-SacII fragment of pTP819.pTP819, in which the escribed . pTP828 cat gene from a Tn9-containing E. coli strain with primers 5'-GACGCACTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGCCGGCCACTGGAGCACCTCAAAAACACCA-3' and 5'-GACGCACTCGAGGCACACAGCGCCCAGCCAACACAGCCAAACATCCGCGCGGGCCCGACCGGGTCGAATTTGCTTTCGAA-3'. The presence of the expected sequences from the synthetic oligonucleotides in pTP921 DNA was verified by automated sequencing (data not shown).pTP921 was constructed by ligating together two XhoI-digested DNAs: pTP922 and a PCR product made by amplifying the lac flank only) was constructed by replacing the XbaI-ApaI lacZY segment of pTP930 with a mixture of two oligonucleotides, 5'-CTAGTTGCAAGCTTGGGCC-3' and 5'-CAAGCTTGCAA-3'. pTP989 (right lac flank only) was constructed by replacing the NgoMI-XhoI lacZ segment of pTP930 with a mixture of two oligonucleotides, 5'-CCGGCAAGCTTGCTGGTGGGCAA-3' and 5'-TCGATTGCCACCAGCAAGCTTG-3'. pTP995 (no lac flank) was constructed by ligating together the small NcoI fragment of pTP988 and the large, ori-containing NcoI fragment of pTP989.pTP988 (left cat-containing fragment of pTP930 and the XbaI- and PaeR7-ended ori-containing fragment of pTP931. pTP1033 was constructed by ligating together the XbaI- and PaeR7-ended cat-containing fragment of pTP931 and the XbaI- and PaeR7-ended ori-containing fragment of pTP930.pTP1032 was constructed by ligating together the XbaI- and PaeR7-ended cat gene and flanking sequences from strain TP872 (Δ lac::cat) with primers 5'-CATCATCACGCGGCCGCGACGTTTGCCGCTTCTGAA-3' and 5'-ATCATCCACGCGGCCGCTGCGTTTTGCACCAGTACG-3'. In pTP1016, the cat gene is flanked closely by unique SphI and BsrGI sites. pTP1027 was constructed by electroporating SphI- and BsrGI-digested pTP1016 into strain TP829 (lac+); a plasmid formed by gap repair was isolated. pTP1028 was constructed by ligating SphI-digested pTP1027 with the oligonucleotide 5'-GTTGTACAACCATG-3', converting the SphI site into a BsrGI site. pTP1034 was constructed by cutting pTP1028 with BsrGI, ligating the two fragments back together, and screening for plasmids in which the lac genes were inverted relative to their flanking sequences.pTP1016 was constructed by ligating into the NotI site of pTP809  the NotIpir function, was constructed by ligating together two AatII- and Bam-digested DNA species: a PCR product made by amplifying a Tn10-containing E. coli strain with primers 5'-TCAACGTAAATGCATGGACGTCCTCGACATCTTGGTTACCGT-3' and 5'-TGTACACCATGAATTGGATCCCGCGGAATAACATCATTTGG-3'; and the ori-containing fragment of plasmid pLD54 [lac::cat-containing fragments of pTP819, 930, 931, 1032, and 1033, respectively, into the BamHI site of pTP1029.pTP1029, a tetracycline resistance bearing vector capable of replicating only in cells expressing R6K id pLD54 . pTP1018cat-containing fragment of pTP1051 into the BamHI site of pTP1029.pTP1047 was constructed by ligating the complementary oligonucleotides 5'-TCGTCTAGAGT-3' and 5'-CCGGACTCTAGACGAAGCT-3' between the SacI and NgoMIV sites of pTP1019. pTP1051 was constructed by ligating the complementary oligonucleotides 5'-CAGCATGCAT-3' and 5'-CTAGATGCATGCTGGTAC-3' between the KpnI and XbaI sites of pTP930. pTP1052 was constructed by ligating the complementary oligonucleotides 5'-CAGCATGCAGGGCC-3' and 5'-CTGCATGCTGGTAC-3' between the KpnI and ApaI sites of pTP1019. pTP1053 was constructed by ligating the complementary oligonucleotides 5'-GGCATGCAGGTTCTTTGAGTCCTTTGGGCGGCCGCGGGCC-3' and 5'-CGCGGCCGCCCAAAGGACTCAAAGAACCTGCATGCCGC-3' between the SacII and ApaI sites of pTP1019. pTP1054 was constructed by ligating the complementary oligonucleotides 5'-CCGGCGCGGCCGCAGGTTCTTTGAGTCCTTTGGTGTACAGAGCT-3' and 5'-CTGTACACCAAAGGACTCAAAGAACCTGCGGCCGCG-3' between the NgoMIV and SacI sites of pTP1020. pTP1055 was constructed by ligating the small SphI-NotI fragment of pTP1016 between the SphI and NotI sites of pTP1053. pTP1056 was constructed by ligating the small NotI-BsrGI fragment of pTP1016 between the NotI and BsrGI sites of pTP1054. pTP1057 was constructed by ligating the BamHI Ptac-gam-bet-exo-pae-cI operon flanked by sequences upstream from recC on one side and sequences internal to recD on the other, has been described [cI gene from E. coli strain TP507 with the same primers used in the construction of pTPP822. This construction has the effect of simply deleting the pae genes from pTP822. pTP996 was constructed by deleting the C-terminal coding sequences of bet and the N-terminal coding sequences of exo between the two HpaI sites of pTP978. pTP979 was constructed by ligating together two NcoI- and XbaI-digested DNAs: pTP822 and a PCR product made by amplifying the bla gene from pBR322 with primers 5'-CCACCAATCATCCATGGCGCGGAACCCCTATTTGTTT-3' and 5'-TTGTTGGACGATCTAGAGGTCTGACAGTTACCAATGC-3'. This construction has the effect of replacing pae and cI with bla.pTP822, which bears a synthetic lac::cat819 and λ lac::cat819 nin5 have been described [nin::tet859 was made by crossing λ wild type with plasmid pTP859, infecting a tetracycline-sensitive strain with the resulting lysate, and selecting a tetracycline-resistant lysogen. The Δ nin::tet859 substitution replaces λ bp 40388–43825 with the tetR and tetA genes of transposon Tn 10.λ escribed . λΔ nin:lac::cat921, 929, 930, 931, 988, 989, 995, 1032, and 1033 substitutions were crossed into λ wild type and/or λ Δ nin::tet859. The parent phages were crossed with the cat substitution-bearing plasmids. Phages which had acquired the substitution were either selected by plating on a strain lysogenic for phage P2 (Spi- phenotype), or identified by their clear-plaque morphologies . The structures of the substituted phages were all verified by their ability to generate specific products when used as templates in PCR (not shown).The 4), and shaken with chloroform. Two methods for crosses monitoring recombination between phage-injected DNA and the bacterial chromosome, both previously described, were employed: high-multiplicity infection of log phase cultures [Phages were crossed with plasmids by spotting enough phage to make a confluent zone of lysis on a lawn of a sensitive bacterial strain bearing the parent plasmid. After overnight incubation at 37°C, material from the zone was collected in TM  dimethylsulfoxide, 20 mM Tris-HCl pH 8.4, 50 mM KCl, and primers at 0.7 μM. Samples were heated to 95C for 5 min, then put through 30 cycles of 94C for 1 min, 55C for 1 min, 72C for 2–4 min, depending upon the length of the expected products. Primers for demonstrating the type I, II, and III tfa junctions were combined in a single mixture of 4 oligonucleotides: Jsp1 (GTTGAATGGGCGGATGCTAA), Jsp2 (TCTTCCACCAGAAAGCTACC), JncA (TGCCGTGTGAACGGTTTAC), and Jnc2 (TTCTAGCCCCATCATCTGTG). Primers for spanning the ACF-generated duplication  and Csj2 (CGAATAGTCGGCTCAACGTGGGTT).The structures of various Red-generated duplications in the PCR: polymerase chain reaction. X-Gal: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. IPTG: isopropyl β-D-thiogalactopyranoside. bp: base pairs.ACF carried out the experiments summarized in Table


HTLV-1 is the etiological agent of adult T-cell leukemia (ATL), the neurological syndrome TSP/HAM and certain other clinical disorders. The viral Tax protein is considered to play a central role in the process leading to ATL. Tax modulates the expression of many viral and cellular genes through the CREB/ATF-, SRF- and NF-κB-associated pathways. In addition, Tax employs the CBP/p300 and p/CAF co-activators for implementing the full transcriptional activation competence of each of these pathways. Tax also affects the function of various other regulatory proteins by direct protein-protein interaction. Through these activities Tax sets the infected T-cells into continuous uncontrolled replication and destabilizes their genome by interfering with the function of telomerase and topoisomerase-I and by inhibiting DNA repair. Furthermore, Tax prevents cell cycle arrest and apoptosis that would otherwise be induced by the unrepaired DNA damage and enables, thereby, accumulation of mutations that can contribute to the leukemogenic process. Together, these capacities render Tax highly oncogenic as reflected by its ability to transform rodent fibroblasts and primary human T-cells and to induce tumors in transgenic mice. In this article we discuss these effects of Tax and their apparent contribution to the HTLV-1 associated leukemogenic process. Notably, however, shortly after infection the virus enters into a latent state, in which viral gene expression is low in most of the HTLV-1 carriers' infected T-cells and so is the level of Tax protein, although rare infected cells may still display high viral RNA. This low Tax level is evidently insufficient for exerting its multiple oncogenic effects. Therefore, we propose that the latent virus must be activated, at least temporarily, in order to elevate Tax to its effective level and that during this transient activation state the infected cells may acquire some oncogenic mutations which can enable them to further progress towards ATL even if the activated virus is re-suppressed after a while. We conclude this review by outlining an hypothetical flow of events from the initial virus infection up to the ultimate ATL development and comment on the risk factors leading to ATL development in some people and to TSP/HAM in others. After binding to the CArG box, SRF protein interacts with the ternary complex factors (TCFs), which consequently bind to the upstream Ets box. In addition, SRF requires for its transcriptional activity the CBP/p300 and p/CAF co-activators [HTLV-1 infected and Tax-expressing T-cell lines display increased expression of immediate early genes such as c-Fos, c-Jun, JunB, JunD and Fra-1, which are components of the dimeric transcription factors AP1, Egr-1 and Egr-2 , fra-1 , Krox-20tivators .Tax activates these immediate early genes by interacting with SRF ,78 and wA substantial part of Tax oncogenic potential is attributed to its ability to activate transcription factors of the NF-κB family, since these factors regulate the expression of numerous cellular genes  associatIn non-activated state NF-κB factors are trapped in the cytoplasm, tightly associated with inhibitory proteins called IκBs, primarily with IκBα and IκBβ. These inhibitors contain ankyrin repeats through which they bind to the RHD of the NF-κB factors and mask their nuclear localization signal (NLS) . In addi on serine19 and serine23  [ share a 52% amino acid identity and a similar domain structure that includes amino-terminal kinase domain, a dimerization leucine zipper domain, and helix-loop-helix motifs, which are involved in regulating their kinase activity [The cytoplasmic phase includes phosphorylation of IκBα on serine32 and serine36 and of IκBr (NEMO) ,90  Fig. , No. 2b,erine181 ,94. Desperine181 ,95,96. Terine181 ,98  can bind the CBP/p300 and P/CAF coactivators which are essential for the transcriptional competence of p65(RelA):p65(RelA) and p65(RelA):p50 dimers . This bi and maintains, in this manner, a persistent NF-κB transcriptional activation [In addition to its cytoplasmic inhibitory function IκBα plays an important regulatory role in the nucleus too. IκBα has an NLS signal which enables its translocation to the nucleus where it is protected from the signal-induced degradation described above . Within tivation . has also been found to have an important role in the nucleus  and IKKα for CBP/p300   The most widely accepted concept is that Tax associates with the IKK complex through the adaptor IKKγ/NEMO subunit. Tax also binds to the upstream kinases, MEKK1 and NIK and enhances their kinase activity. In this manner Tax connects these activated kinases to IKKγ/NEMO and recruits their kinase activity to phosphorylate IKK and IKKβ [b) Tax can bind directly to IKK and IKK and activates their kinase activity independently of their phosphorylation by the upstream signal-induced kinases [c) Tax can bind directly to the IκBs and induce their proteosomal degradation independently of their phosphorylation by IKK [In contrast to the transient NF-κB activation by external signals, NF-κB factors are constitutively activated by HTLV-1 Tax protein in Tax-expressing and HTLV-1-infected cells. Reported studies suggest that Tax may exert this activation in three ways: and IKKβ ,120-122 and IKKβ . b) Tax  kinases  Fig. 3,,a The mon by IKK ,125  is involved in DNA synthesis and maintenance of the genome stability by participating in DNA repair and chromosome condensation. It alters DNA topology by transiently breaking one strand of the DNA, passing the other strand through the break and finally resealing the break . Tax hasAll the above effects of Tax which enhance mutations and other chromosomal aberrations and interfere with DNA repair should be expected to induce cell cycle arrest or apoptosis. This, in turn, should prevent further progression of the leukemogenic process in the infected cells, unless such cells can, somehow, escape the cell cycle arrest and apoptosis. There is a substantial controversy over the influence of Tax on the cell response to stress insults. While many studies have demonstrated that Tax protects cells from stress-induced cell cycle arrest or apoptosis -186, othNumerous studies have been focused on investigating Tax oncogenic potential in cultured cells and animal models. Most of them used plasmids expressing w.t. Tax or various Tax mutants under the control of HTLV-1 LTR or other different promoters. It was only few years ago that an infectious clone of the entire HTLV-1 genome was constructed and appropriate cell culture techniques were developed to introduce this clone into human primary PBLs. This clone was found as capable of propagating in cultured mammalian cells and to transform primary human T-lymphocytes -197. ThiWAF-1 which prevents apoptosis and enhances the replication of the transformed cells [Tax has been shown to induce neoplastic transformation of the rat fibroblast Rat-1 -203 and ed cells . The cooed cells . It shoued cells . Therefoed cells ,153,165.WAF-1 [A closer insight into the ATL leukemogenesis has been gained through studies using primary human T-lymphocytes. Such experiments have revealed that after infection in culture with HTLV-1 or permanent transfection with Tax, primary human T-cells undergo two stages of cellular changes. In the first stage the cell become immortalized but still remain dependent on IL-2 for their growth . This imWAF-1  and to bWAF-1 . StudiesWAF-1 ,208, othWAF-1 . On the WAF-1 . In addiWAF-1 ,210.In the second stage few IL-2-independent clones of transformed cell emerge. Such transformed cells display an IL-2-independent constitutive activation of the IL-2 receptor (IL-2R) signaling pathway that includes the Janus kinases JAK1 and JAK3, and the signal transducers and activators of transcription STAT3 and STAT5, which are constitutively active in such cells ,211-213.Many of the above changes observed in the HTLV-1/Tax immortalized and transformed primary human T-cells are quite analogous to those found in ATL cells. However, while fresh leukemic cells from ATL patients, as well as cell lines derived from these leukemic cells, are successfully engrafted in SCID mice and their leukemic infiltration to various organs is similar to that seen in ATL patients , primaryTax transgenic mice have been widely used as models for investigating the oncogenic effects of Tax in-vivo, hoping to get closer insight to the ATL leukemogenic process in human . A wide Various modes of tumor induction by the transgenic Tax have been noted so far. Hall et al.  have shoAs noted before, continuous Tax expression is required for maintaining the neoplastic phenotype of Rat-1 cells transformed by Tax in culture . In contINK4B and p16INK4A [KIP1 [In view of the above described pleiotropic effects of Tax, which are summarized in Fig. p16INK4A , p27KIP14A [KIP1 , p16  prepared the Fig.ures and together with author 2 (S-K.Y) covered the cited publications and prepared the draft of this review. Author 3 (A.M) designed the outlines of the review and together with the other two authors prepared the final version for submission. All authors read and approved the final manuscript.


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 . 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  and peanut was more commonly reported by males .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  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 . 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  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  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  per μg RNA. Two to 2.5 μg total RNA was subjected to first strand cDNA synthesis using SuperScript™ II  reagents in a 20 μl volume. DNA was extracted from the cells using GenElute™ Mammalian Genomic DNA Kit  and stored at -20°C until use.Human Umbilical Vein Endothelial Cells (HUVEC) and Human Aortic Endothelial Cells (HAEC)  were grown in Medium 200 with Low Serum Growth Supplement  at 37°C in a humidified atmosphere of 5% CO® Gold DNA polymerase , 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 . The amplified fragments were combined and concentrated to 60 μl using Microcon ® YM-30 Centrifugal Filter Devices .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  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  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  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 .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  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 . 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.