Arabidopsis genome revealed that Pseudoviridae insert randomly along the chromosome and have been recently active whereas Metaviridae were more active in the past and preferentially target heterochromatin.An analysis of the distribution and age of LTR retrotransposons in the  Genome evolution and size variation in multicellular organisms are profoundly influenced by the activity of retrotransposons. In higher eukaryotes with compact genomes retrotransposons are found in lower copy numbers than in larger genomes, which could be due to either suppression of transposition or to elimination of insertions, and are non-randomly distributed along the chromosomes. The evolutionary mechanisms constraining retrotransposon copy number and chromosomal distribution are still poorly understood.Arabidopsis thaliana genome, using an automated method for obtaining genome-wide, age and physical distribution profiles for different groups of elements, and then comparing the distributions of young and old insertions. Elements of the Pseudoviridae family insert randomly along the chromosomes and have been recently active, but insertions tend to be lost from euchromatic regions where they are less likely to fix, with a half-life estimated at approximately 470,000 years. In contrast, members of the Metaviridae (particularly Athila) preferentially target heterochromatin, and were more active in the past.I investigated the evolutionary dynamics of long terminal repeat (LTR)-retrotransposons in the compact A. thaliana, their non-random genomic distribution is due to both selection against insertions in euchromatin and preferential targeting of heterochromatin. Constant turnover of euchromatic insertions and a decline in activity for the elements that target heterochromatin have both limited the contribution of retrotransposon DNA to genome size expansion in A. thaliana.Diverse evolutionary mechanisms have constrained both the copy number and chromosomal distribution of retrotransposons within a single genome. In  Drosophila melanogaster [Ty1/Copia elements) and Metaviridae (Ty3/Gypsy).It has become increasingly clear that the activity of transposable elements (TEs) is a major cause of genome evolution. TEs are ubiquitous components of eukaryotic genomes. For example, 22% of the nogaster , 45% of nogaster  genomes nogaster . In plannogaster  and/or anogaster ,8-10. LTThe evolutionary forces that control copy number and shape the chromosomal distribution of different kinds of TEs in eukaryotic genomes are still poorly understood. Some large plant and animal genomes have expanded owing to an ability to tolerate massive amplification of retrotransposons, whereas in more compact genomes these elements are found in lower copy numbers, non-randomly distributed and mainly confined to heterochromatic regions -14. TEs It is often hypothesized that the non-random genomic distribution of TEs in some species reflects the action of purifying selection on the host against the deleterious effects of TE insertions in certain regions. Models differ in the kind of deleterious effects they propose: chromosomal rearrangements due to 'ectopic'  recombination ; disruptArabidopsis thaliana genome. The total TE content of the compact genome of A. thaliana, with a haploid size of approximately 150 Mbp (million base-pairs), has been previously estimated as around 10%, and is known to cluster around the pericentromeric heterochromatin [A. thaliana [A. thaliana. By comparing the age and chromosomal distributions of young and old insertions it is possible to distinguish between preferential targeting and passive accumulation of elements into heterochromatin. I show that members of the Pseudoviridae have recently been active, that they integrate randomly into the genome (relative to centromere location) and only passively accumulate in proximal regions, as purifying selection eliminates euchromatic insertions. In contrast, the Metaviridae (particularly members of the Athila group) preferentially insert into the pericentromeric heterochromatin, and their transpositional activity has declined in the last million years.To evaluate these hypotheses, I investigated the evolutionary history of different groups of LTR-RTs in the hromatin . Despitethaliana ,23. I haAthila superfamily, though the core centromere sequences themselves were not available. In fact, the size of the A. thaliana genome has been recently estimated as approximately 157 Mbp (around 20% larger than the estimate published with the genome sequence), and the additional size appears to be due to (unsequenced) heterochromatic repetitive DNA in the centromeres, telomeres and nucleolar-organizing regions [Athila is the most abundant superfamily, followed by the Copia-like, Gypsy-like, and TRIM . The ratio of solo-LTRs to complete elements is around 2:1. In addition to solo-LTR formation, deletion and fragmentation of retrotransposon DNA in A. thaliana also occur via other mechanisms: 36% of the DNA in the Athila, 38% in the Gypsy-like, 32% in the Copia-like, and 21% in the TRIM superfamilies correspond to degraded insertions that are neither 'complete' elements nor solo-LTRs.Most of the retrieved elements are fragmented and truncated, and nested insertions are common particularly among pericentromeric elements belonging to the  regions .  Table Katydid-At1 and Katydid-At2, most intra-element LTR pairs did not cluster. In contrast, when trees were constructed for representatives of the Athila (athila2), Gypsy-like (atlantys2), and Copia-like (meta1, atcopia49, atcopia78) superfamilies, intra-element LTR pairs always clustered (data not shown), providing evidence for the lack of inter-element recombination in those 'families'.To obtain the genome-wide age distribution of each superfamily (except TRIM), 564 pairs of intra-element LTRs were (pairwise) aligned and their sequence divergence estimated. Many of the complete TRIM elements have highly divergent LTRs, and I suspect that extensive recombination between inter-element LTRs has occurred. In neighbor-joining trees of LTR sequences (of both complete and solo elements) from the TRIM families Athila insertions are significantly older than the Gypsy-like (Wilcoxon rank-sum test, p < 0.0005), Gypsy-like older than Copia-like (p < 0.0001). Age distributions are summarized in Figure The superfamilies differ significantly in their average age of insertions. p < 0.000. Age dis-8 substitutions per site per year [Copia-like elements are younger than 3 million years (Myr), 90% younger than 2 Myr, and only two insertions estimated to be older than 4 Myr. This shows that complete insertions from the known Copia-like families in the A. thaliana genome are younger than the species itself, whose time of divergence from its closest relatives, such as A. lyrata has been estimated (with the same rate of evolution) to be 5.1-5.4 Myr ago [Athila (and the Gypsy-like TEs), as 7% of 219 intra-element LTR pairs were estimated to be older than 5 Myr (3% of the Gypsy-like). Furthermore, the Athila and Gypsy-like superfamilies have an excess of degraded insertions relative to Copia-like (Table Athila and Gypsy-like, indicating that the majority of insertions remaining in the genome have been degraded or have become solo-LTRs. Some of these are likely to be older than the complete insertions. DNA loss (from LTR-RTs) has been shown to occur in A. thaliana [Arabidopsis are not evolving in a completely neutral fashion [Chs) and alcohol dehydrogenase (Adh) loci, their synonymous sites would be evolving more slowly than LTR-RT fossils, and the dating method described above would systematically overestimate the ages of their insertion events.Using the rate of 1.5 × 10per year , 97% of  Myr ago . The sitthaliana , and the fashion . If thisCopia-like elements appears to show a recent burst of activity  in  regions . HoweverAthila elements are almost exclusively inserted in the pericentromeric regions, and the other superfamilies in significantly and progressively less proximal regions of the chromosome arms . Furthermore, except for TRIM, within each superfamily the solo-LTRs are significantly more distal than the complete elements (Wilcoxon rank sum tests, p < 0.001), suggesting that formation of solo-LTRs is more likely to occur in distal regions. The distribution of complete TRIM elements relative to the centromere is not significantly different from random (goodness-of-fit test, χ2 = 4.22, df = 3, p > 0.2), although sample size is small, while their solo-LTRs are significantly clustered (goodness-of-fit test, χ2 = 10.70, df = 3, p < 0.02).A. thaliana would affect age comparisons among different groups of elements, as they show different degrees of clustering into the pericentromeric heterochromatin. However, older estimates for the age of heterochromatic elements are consistent with the hypothesis that heterochromatin is a 'safe haven' where TE insertions persist for longer periods of time. Here I show that the mechanisms that led to the accumulation of LTR-RTs in proximal regions are distinct for different groups: elements of the youngest superfamily (Copia-like) insert randomly into the genome (relative to the location of the pericentromeric heterochromatin), but there is negative selection (on the host genome) against their insertions in euchromatin; elements of the older superfamilies (Athila, Gypsy-like) preferentially insert into the pericentromeric regions. These distinct mechanisms become apparent when temporal and spatial data are combined  regions are more likely to persist for longer periods of time. This interpretation assumes that the neutral mutation rate is the same for both the distal (euchromatic) and proximal (heterochromatic) portions of the genome. Intra-genomic variation in the per-replication mutation rate has been reported between the two sex chromosomes of a flowering plant  . Furthermore, the distribution along the chromosome arms of 34 Copia-like insertions with no divergence between their intra-element LTRs is not significantly different from random (goodness-of-fit test, χ2 = 3.12, df = 3, p > 0.3). This is evidence that Copia-like elements integrate randomly relative to the location of the centromeres, but tend to get eliminated from distal, and passively accumulate in proximal regions.For complete t) for a neutral allele is given by t = 4Ne, where Ne is the effective population size. For A. thaliana t can be estimated using an average of estimates of nucleotide diversity (θ) for 8 different A. thaliana genes, θ = 9 × 10-3 [μ = 1.5 × 10-8 [t = 2θ/μ, yielding an estimate of t ≈ 1.2 Myr. This value for t is consistent with an independent estimate that placed the time since the divergence between A. thaliana and A. lyrata between 3.45t and 5.6t [Copia-like insertions are younger than 1.2 Myr, most of them are likely to be polymorphic. Taken together with the highly significant negative correlation between age and distance from the pericentromeric regions, these results indicate that complete Copia-like insertions are less likely to get fixed in the distal, euchromatic portions of the chromosome arms than in the pericentromeric heterochromatin.The average time to fixation (9 × 10-3 , and the5 × 10-8 . t = 2θ/and 5.6t . Given tAthila elements (Spearman rank correlation, ρ = 0.01, p = 0.9), as both young and old insertions are found only in proximal regions . If the arm is excluded from the analysis there is no significant correlation (Spearman rank correlation, ρ = -0.09, p > 0.3). This suggests that for the Gypsy-like also there is an insertional bias towards proximal regions. This bias is not as strong as for Athila, as complete Gypsy-like insertions are not exclusively found around the centromeres, and they cluster (to a much lesser extent) in at least one other heterochromatic region (the telomere of the short arm of chromosome II). Included in the Gypsy-like 'superfamily' is a clade of elements, known as Tat, which is a sister group to Athila to the exclusion of the remaining Gypsy-like elements [Tat does not differ from those of the remaining Gypsy-like elements (Wilcoxon rank-sum tests, p > 0.4); Tat show insertion bias towards the pericentromeric regions, but again to a lesser degree than Athila.elements . The ageCopia-like elements have been active until recently but tend to be eliminated by purifying selection, their age distribution  = NoeaK-, where N(K) is the number of elements observed with intra-element LTR divergence K, and No and a are constants to be fitted. The rate of elimination can then be estimated by linear regression of the log-transformed data . Figure Copia-like insertions (R2 = 0.94), and for complete insertions outside the proximal regions . Complete Copia-like elements are eliminated from the genome with a half-life of 648,000 years (SE = 48,000 years). Insertions exclusively outside the proximal (heterochromatic) regions are lost more rapidly, with a half-life of 472,000 years (SE = 46,000 years).Given that n Figure , bottom A. thaliana the accumulation of insertions in the pericentromeric heterochromatin is the result of both insertion bias (for Metaviridae elements) and a lower probability of fixation in euchromatin (Pseudoviridae).The results above indicate that within a single genome, distinct evolutionary mechanisms can lead to the non-random distribution of retrotransposons, as in A. thaliana were already present in its common ancestor with Brassica oleracea (the two species diverged around 15-20 Myr ago), and that copy numbers are generally higher in B. oleracea [A. thaliana and B. oleracea was responsible for the larger genome of the latter. Here I have shown that the major LTR-RT families have been active in A. thaliana since its divergence from its closest relatives, such as A. lyrata. The transpositional activity of Metaviridae elements has declined relative to its level between 1 and 2 Myr ago, perhaps suggesting that the host genome has more efficiently suppressed their transposition since. However, Pseudoviridae (Copia-like) elements in A. thaliana have been subject to constant turnover. They have been recently active and show no insertion bias, and I estimate that the half-life of a complete element inserted in the euchromatic (non-coding) regions of the chromosome arms as around 470,000 years. Most of these Pseudoviridae insertions are lost before they reach fixation, and the half-life estimate provides a measure of the pace at which natural selection on the host constrains the genomic distribution and copy number of Pseudoviridae insertions. Turnover of Pseudoviridae insertions, in contrast to the longer persistence of Metaviridae elements that have declined in activity, is consistent with the higher sequence diversity among the Pseudoviridae than the Metaviridae in A. thaliana (107 Repbase update (RU) 'families' represented in 215 complete Pseudoviridaeelements, 25 RU 'families' in 349 complete Metaviridae elements, where 'families' were defined on the basis of sequence divergence); frequent reverse transcription during transposition would be likely to lead to faster evolution than that generated by the host genome DNA polymerase error rate on chromosomal insertions.It has recently been shown that most TE lineages in oleracea . The autA. thaliana genome does not correlate with local recombination rate [The lower probability of fixation in euchromatin relative to heterochromatin implies that insertions into euchromatin are more deleterious to the host (and perhaps that purifying selection is less efficient in heterochromatin due to a much reduced rate of recombination). TE density in the ion rate , providiion rate .Copia-like elements, if such a process occurred more often in the euchromatic than in the pericentromeric regions of the genome, and if it were frequent enough. One mechanism that removes LTR-RT DNA from the genome is solo-LTR formation via unequal homologous recombination between intra-element LTRs. However, this mechanism cannot be the driving force shaping the distribution of complete Copia-like elements because Copia-like solo-LTRs are also non-randomly distributed and clustered in proximal regions (goodness-of-fit test: χ2 = 13.71, df = 3, p < 0.005). Copia-like solo-LTRs are either eliminated faster from distal than proximal regions, like complete elements, or solo-LTR formation on average occurs more slowly than extinction for euchromatic insertions. Despite clustering around the centromeres, Copia-like solo-LTRs are significantly more dispersed than complete elements. This suggests that solo-LTRs do form before extinction for distal insertions, but are probably less efficiently eliminated (possibly because they are less deleterious to the host genome) than complete elements. Another known mechanism of  DNA loss operates via small deletions due to illegitimate recombination (between short repeats); this has been shown to occur in the A. thaliana genome by an analysis of internal deletions in LTR-RTs [Drosophila, rates of spontaneous deletions in euchromatin and heterochromatin do not seem to differ [A. thaliana the relative rates between the two chromatin domains are unknown, but fragmented (that is, neither solo-LTR nor complete) Copia-like insertions are as clustered around the centromeres as complete ones (goodness-of-fit test: χ2 = 80.36, df = 3, p < 0.0001). Therefore small, spontaneous deletions cannot account for the genomic distribution of complete elements. Larger deletions (that remove the entire LTR-RT sequence) occurring primarily in euchromatin would be necessary to explain the observed accumulation pattern; if such a mechanism existed it would be an important force for genome size contraction. As there is no evidence for such mechanism, and given that I estimate that the half-life of (complete) insertions to be less than half the average time to fixation for a neutral allele, a lower probability of fixation in euchromatin relative to the pericentromeric heterochromatin is more likely to be driving the genomic distribution of Pseudoviridae elements.As an alternative to selection, a neutral mutational process that deletes (part of the) insertions could in principle be driving the distribution of  LTR-RTs . In Droso differ . In A. tAthila elements have been known to be present in the A. thaliana core centromeric arrays of the 180-bp satellite repeats and are abundant in pericentromeric heterochromatin [Copia-like elements, the striking compartmentalization of both recent and older Athila insertions in the pericentromeric heterochromatin indicates that these elements actively target those regions, and represents an example of a group of retrotransposons that have evolved to colonize a particular 'genomic niche'. Passive accumulation could not explain the distribution of Athila insertions unless they were generally much more deleterious to their host than Copia-like ones. Given the absence of complete Athila insertions from euchromatin, any one insertion would have to be so deleterious as to be almost immediately eliminated by purifying selection, even from intergenic DNA. Rather, it is likely that Athila elements preferentially insert into the pericentromeric heterochromatin and it is possible that this group of elements has been co-opted to play a part in centromere function. There is some evidence that such hypothetical role cannot be that of cis-acting sequences [A. thaliana [It is interesting to note that the integrase proteins encoded by LTR-RTs differ between the Pseudoviridae and the Metaviridae in their carboxy-terminal domains, as they have different characteristic motifs ,36. Thishromatin ,41. In tequences , but it equences -45 pointequences , centromequences -51, chicequences  and A. tthaliana .A. thaliana. The rapid and recent genome evolution inferred for A. thaliana may be a feature common to other higher eukaryotes, in particular those with compact genomes. High turnover of TE insertions in euchromatin also occurs in Drosophila and pufferfish [A. thaliana, be due to diverse evolutionary mechanisms.Both purifying selection (at the host level) against insertions (in euchromatin) and a decline in transpositional activity (of Metaviridae elements) appear to have limited the recent contribution of retrotransposon DNA to genome size expansion in fferfish , for exaA. thaliana.A methodology was developed for the automated mining of sequence data to retrieve the sequence and chromosomal location of genomic 'fossils' of LTR-RTs, identifying complete elements and solo-LTRs among the retrieved sequence fragments, and estimating the age of the insertion events that gave origin to these elements. This methodology was applied to the genome sequence of A. thaliana genome (around 116 Mbp of available sequence) was performed using sequence-similarity search algorithms [A. thaliana 'families' of LTR-RTs. The programs RepeatMasker [Sequences of the organellar and the five nuclear chromosomes (version 200303) were obtained from the Munich Information Center for Protein Sequences (MIPS) . Computagorithms  against gorithms ,57 sequeatMasker  and WU-BatMasker  were useAthila, Gypsy-like , and Copia-like . The Metaviridae was split into two groups (Athila and Gypsy-like), as initial mining of the A. thaliana genome revealed that Athila elements have been particularly successful in colonizing it. Their copy number is roughly double the number of all other members of the Metaviridae, and higher than the total of all Pseudoviridae elements. Athila form a clade and are retroviral-like elements that are likely to have an envelope (env) gene [Copia- and Gypsy-like elements are typical LTR-RTs, although one of the Copia-like 'families' (metaI) comprises non-autonomous elements [endovir1 [atcopia41-43 [env gene. A fourth 'superfamily' was used to include TRIMs. These are short, non-autonomous elements that feature LTRs but no coding genes and cannot currently be classified into either the Pseudoviridae or the Metaviridae; they are described in ['Families' of LTR-retrotransposons (as classified in Repbase update) are present in low copy numbers; therefore, for the purpose of this analysis they were grouped into three 'superfamilies': nv) gene . Most ofelements  and a feendovir1 , atcopiapia41-43 ) are retribed in .Athila (10 families): athila2 - 5, athila4A - D, athila6A, athila7, athila8A and B; Gypsy-like (15 families): atgagpol1, atgp2 and 3, atgp2N, atgp5 - 10, atgp9B, atlantys1 - 3, tat1; Copia-like (107 families): atcopia1 - 97, atcopia8A and B, atcopia18A, atcopia32B, atcopia38A and B, atcopia65A, endovir1, TA1-2, meta1; TRIM (3 families): katydid-At1, katydid-At2, katydid-At3.The four superfamilies comprise the following 'families'. Perl script, LTR_MINER (available on request), was written to parse all the chromosomal LTR-RT fragments reported by RepeatMasker (WU-BLAST hits of similarity to reference sequences) and identify complete elements and solo-LTRs. LTR_MINER performs the pattern-recognition function of assembling hits that originated from single LTR-RT insertion events. The algorithm involves: 'defragmentation' of LTR hits. If a chromosomal LTR fossil contains insertions/deletions (indels) relative to the most similar library sequence, it may be reported as multiple hits (fragments). Defragmentation is the identification of multiple hits that correspond to the same LTR. Parameters were set so that LTR hits were defragmented only when they were separated by no more than 550 bp, belonged to the same family, had the same orientation on the chromosome, and their combined length did not exceed the length of the corresponding family reference sequence by more than 20 bp.A LTR, IR, LTR. In order to check whether the pattern could be straddling a nested insertion of the same family, the search is then recursively extended from each end of the pattern for further contiguous hits to an IR and a LTR (of same family and orientation). The two LTRs of the innermost pattern are classified as a pair of intra-element LTRs.An intact LTR-RT insertion consists of at least three hits: LTR-IR-LTR . After LTR defragmentation, LTR_MINER searches for contiguous patterns of LTR_MINER also identifies such elements provided an IR is present between the LTRs. A maximum pairing distance between LTRs was set at 30 kb.LTR_MINER was set to classify a LTR fragment as a solo-LTR if no other LTR or IR (of same family and orientation) is present within a 5 kbp radius from the fragment's ends. The aim was the identification of elements resulting from deletion (of the IR and one LTR) events via homologous recombination between intra-element LTRs, and not to classify as solo-LTRs sequences that are separated from IRs because of insertions.K) between each intra-element LTR pair was then estimated using Kimura's two-parameter model [Nucleotide sequence divergence between pairs of intra-element LTRs was used as a molecular clock, as these pairs are identical at the time of insertion . All miner model . To reduK greater than 0.2 they were inspected by eye and manually edited if necessary . Estimates of the ages of insertion were obtained by using the equation t = K/2r, where t is the age, and r is nucleotide substitution rate for the host genome DNA polymerase. The value of 1.5 × 10-8 substitutions per site per year was used for r , estimated in [Chs and Adh loci in Arabidopsis/Arabis species.Alignments with fewer than 80 nucleotides were discarded. As CLUSTAL-W alignments could be poor if LTR sequences were only partially overlapping, for all LTR pairs with mated in  for the Finally, if recombination between LTRs from different insertions had occurred frequently, the dating method above would be invalid for obtaining the age profiles of different families. To detect possible recombination events, multiple alignments of all LTRs (including solos) of certain families were generated using BLASTALIGN , a progrA. thaliana. Additional data file A. thaliana. Additional data file Perl script LTR_MINER, used to de-fragment sequence similarity hits to LTR-retrotransposons, and identify complete and solo-LTR elements. Additional data file Perl script in Additional data file Perl script used in conjunction with LTR_MINER, used to divide long sequences into smaller chunks labeled by their coordinate range. Additional file data Perl script in Additional data file The following additional data files are available with the online version of this article. Additional data file A. thalianaThe entire dataset of chromosomal coordinates and ages of complete LTR-retrotransposons in Click here for additional data fileA file describing the data fields in Additional data file 1Click here for additional data fileA. thalianaThe entire dataset of chromosomal coordinates of solo-LTRs in Click here for additional data fileA file describing the data fields in Additional data file 3Click here for additional data filePerl script LTR_MINER, used to de-fragment sequence similarity hits to LTR-retrotransposons, and identify complete and solo-LTR elementsThe Click here for additional data filePerl script in Additional data file 5A file describing the utility and usage of the Click here for additional data filePerl script used in conjunction with LTR_MINER, used to divide long sequences into smaller chunks labeled by their coordinate rangeThe Click here for additional data filePerl script in Additional data file 7A file describing the utility and usage of the Click here for additional data file


Epidemiological studies have found reduced health-related quality of life (QoL) in patients with personality disorders (PDs), but few clinical studies have examined QoL in PDs, and none of them are from an ordinary psychiatric outpatient clinic (POC). We wanted to examine QoL in patients with PDs seen at a POC, to explore the associations of QoL with established psychiatric measures, and to evaluate QoL as an outcome measure in PD patients.72 patients with PDs at a POC filled in the MOS Short Form 36 (SF-36), and two established psychiatric self-rating measures. A national norm sample was compared on the SF-36. An independent psychiatrist diagnosed PDs and Axis-I disorders by structured interviews and rated the Global Assessment of Functioning (GAF). All measurements were repeated in the 39 PD patients that attended the 2 years follow-up examination.PD patients showed high co-morbidity with other PDs and Axis I mental disorders, and they scored significantly lower on all the SF-36 dimensions than age- and gender-adjusted norms. Adjustment for co-morbid Axis I disorders had some influence, however. The SF-36 mental health, vitality, and social functioning were significantly associated with the GAF and the self-rated psychiatric measures. Significant changes at follow-up were found in the psychiatric measures, but only on the mental health and role-physical of the SF-36.Patients with PDs seen for treatment at a POC have globally poor QoL. Both physical and mental dimensions of the SF-36 are correlated with established psychiatric measures in such patients, but significant changes in these measures are only partly associated with changes in the SF-36 dimensions. According to the DSM-IV  personalSince the DSM-IV included the Global Assessment of Functioning Scale (GAF) as Axis V, it is reasonable to consider if the GAF should be used for evaluations of "significant distress or impairment". Two problems are implicit in such an approach: at what GAF cut-off score should "significant distress or impairment" be set, and the evaluation is only done by a professional. As to the first problem, Kessler et al  suggesteMajor treatment outcome studies of PDs like the Collaborative Longitudinal Personality Disorders Study , the NorIn contrast, two epidemiological studies of PDs have used the SF-12 as a measure of disability. In a national study from Australia, Jackson and Burgess  reportedA review of the literature showed that the QoL had only been used as a disability measure in a few clinical research studies of PD patients. In depressed elderly patients, Abrams et al  found thNone of these clinical studies takes QoL as observed in PD patients seen at an ordinary psychiatric outpatient clinic (POC) as their point of departure. However PD patients are frequent at POC, and the QoL is an important self-rated measure of "clinically significant distress or impairment". Since QoL data on PD patients seen at at POC seems to be lacking from the literature, we found it relevant to study a consecutive sample of PD patients from a POC and collect QoL data with SF-36. We posed the following research questions: 1) How are the SF-36 dimensions mean scores in PD patients compared to age- and gender-adjusted norm data? 2) To what extent are the SF-36 scores in PD patients associated with co-morbid Axis I disorders? 3) How is the association between the SF-36 dimension and established patient- and professional-rated psychiatric measures in PD patients? and 4) What changes in the SF-36 dimensions of treated PD patients are observed from baseline to follow-up, and how are they related to changes in the psychiatric measures?Furuset POC serves a communality of Oslo City, Norway with a population of 28.000 people. At the time of the study, the staff consisted of three psychiatrists, three clinical psychologists, two psychiatric nurses, and two social workers. The intake rate was approximately 400 new patients a year. The first author (KN) invited the staff to take part in the study by referring to her new patients with probable PDs. Six professionals were willing to participate, while four declined due to heavy clinical burden, or lack of interest.At the start of the study in 1996, Furuset was a new suburb of Oslo, and the inhabitants were characterized by lower socio-economic conditions, high mobility, and a considerable prevalence of immigrants from Asian countries. The suburb had a high proportion of municipal housings, and the criterion for allotment to them was severe mental disorder and/or severe socio-economic problems. Many patients seen at Furuset POC were out of work due to mental disorders, and/or due to socio-economic circumstances.Patients aged from 18 to 75 years were consecutively recruited from January 1, 1996 to June 30, 1998. The patients were referred from the local GPs, and physical examination and adequate treatment and follow-up of physical diseases were the responsibility of the GPs. The six therapists screened for probable PDs among new patients scheduled for treatment. Exclusion criteria were mental retardation, lifetime psychosis and bipolar disorder, organic mental disorders, current strong suicidal ideation, and insufficient knowledge of the Norwegian language. Eligible patients received oral and written information about the study from their therapists. Then the patients were invited to take part in the study, and they all gave written informed consent. The Ethical Review Board of Department of Psychiatry, Aker University Hospital approved the project.The six therapists did not miss out any patients at screening, but 5 (4%) eligible patients declined to take part in the study. Among 110 eligible patients referred to the study, only 91 filled in the SF-36 at baseline due to administrative misunderstandings. However, when they were compared to the 19 who did not fill in, the non-attenders only had significantly fewer co-morbid Axis I-disorders (data not shown).In order to answer the research questions, the sample was divided into three groups: cluster A+B PDs (n = 39), cluster C (n = 33), and Axis I-disorders (n = 19). The cluster A+B group could also contain co-morbid cluster C PDs and Axis I-disorders, and the cluster C co-morbid Axis I-disorders.Two years after baseline, the patients received a mailed written appointment for a follow-up interview. Those who did not show up were sent a written reminder. If they still did not meet, they were called by phone, and if there was no answer, their addresses and phone numbers were checked at the Census register. Appointments were mailed to new addresses, and phone-calls were made in case of non-response. Only a few patients responded to these extended search procedures.Norm data on the SF-36 was obtained from the Survey of Level of Living in Norway 1998  comprisiAt baseline, diagnoses of PDs were made with the use of the Personality Disorder Examination, and Axis I-disorders were diagnosed by the MINI-International Neuropsychiatric Interview. Anamnestic data were collected, and global assessment of function was rated. The professional-based interviews and examinations of all patients at baseline and follow-up were carried out by a single experienced psychiatrist (KN), who did not take part in any treatment given.All patients also filled in the following self-rating instruments at baseline: the SF-36, the Social Adjustment Scale, and the Symptom Checklist 90-Revised Personality Severity Index.At follow-up all these assessments were repeated, and additional information about treatment as well as job/education, social- and family changes was collected.Personality Disorder Examination (PDE) [The on (PDE)  is a strMINI International Neuropsychiatric Interview [The nterview  was usedGlobal Assessment of Functioning (GAF) is a rating scale for the current evaluation of the overall functioning of a subject on a continuum from severe mental disorder to complete mental health that was defined as Axis V of the DSM-IV. Scale values range from 1  to 100 . The scale is divided in ten equal intervals from 1 – 10 to 91 – 100. Most outpatients will be rated between 40 and 70, although some individuals rated above 70 may seek therapy. The GAF is a reliable instrument [The strument , and thestrument  and for strument .SF-36 [The SF-36  was chosSF-36 . The SF-Social Adjustment Scale (SAS-SR) [The (SAS-SR)  containsSymptom Checklist 90-Revised (SCL-90-R), Personality Severity Index (PSI). The SCL-90-R is a 90-item self-report inventory assessing current levels of mental symptoms patterns. Each item is a description of a mental symptom rated on a five-point scale, and rates the degree of 'distress/discomfort' during the last week prior to its administration. Several indices based on the SCL-90-R scores have been defined, and the PSI is the mean value of 22 items covering the interpersonal sensitivity, anger/ hostility, and paranoid ideation subscales. The PSI reflects the presence and severity of relatively enduring characteristics of the patient, and is, therefore, relevant for the evaluation of severe PDs [The vere PDs . For the2 and Fisher's Exact Test for categorical variables. The Mann-Whitney test and the Wilcoxon signed-rank test were applied for metric variables when the data distribution violated parametric assumptions. Spearman's rank correlation was used for associations. One-sample t-tests were applied when the SF-36 dimension scores of the age- and gender-adjusted norm groups were compared to the means of the three diagnostic groups. The comparison of mean scores on the SF-36 dimensions between the three groups was conducted with oneway ANOVA with Bonferroni's correction. The influence on the QoL scores of patients with PDs of Axis I disorders and of cluster C PDs in the cluster A+B PDs group, was examined with linear regression analyses. All tests were two-sided, and the level of significance was set at p < .05.Data were analyzed by SPSS version 12.0. Descriptive statistics were conducted with independent and paired-sample t-test as well as one-way ANOVA (with Bonferroni's correction for multiple comparisons) for metric variables, and with χAll the 91 patients included were Caucasian, and 48 (53%) were females. The mean age of the sample at baseline was 36.3 years (SD 10.5) and ranging from 19 to 74 years, with a median of 35 years. None of the patients had any significant somatic diseases as reported by their GPs.Patients belonging to the cluster A+B, cluster C, and Axis I- disorders groups did not differ at baseline on demographic variables Table .As to psychopathology, the Cluster A and B PDs criteria sum scores were significantly higher in the Cluster A+B group compared to the two other groups, and both cluster A+B and cluster C group had significantly higher scores on cluster C criteria sum score than the Axis I group Table . The CluThe diagnostic distribution of PDs and Axis I disorders in the three groups at baseline and follow-up are given in Table Axis-I disorders were equally common in the two PD groups (mean 1.5 disorder) and with slightly lower mean (1.2) in the Axis I-disorder group. Depressions, anxiety disorders, and alcohol dependence were the most frequent Axis I diagnoses in all groups.Figure Linear regression analyses showed that control for co-morbid Axis I disorders reduced the PF, GH, VT, SF, and MH scores of the total PD group significantly. Controlling for comorbid cluster C PDs did not influence the SF-36 scores of the cluster A+B PDs to any significant extent, while control for Axis disorders significantly reduced the MH scores in the cluster A+B and cluster C groups.No significant differences were found between genders on any of the SF-36 dimensions among the PD patients (data not shown). Both the cluster A+B, the cluster C, and the Axis I group differed significantly from their norms on all eight SF-36 dimensions (data not shown). No significant differences were observed on the eight SF-36 dimensions between the three diagnostic groups Figure .When we compared the patients with one (n = 37), two (n = 20), and three or more (n = 15) PDs, we did not observe any significant differences in mean MCS and PCS scores.The eight dimensions of SF-36 are regularly divided into the four physical: PF, RP, BP, and GH, and the four mental ones: VT, SF, RE, and MH. In our PD sample the SF-36 mental dimensions had most significant correlations with the psychiatric measures of the GAF, the SCL-90-R PSI, the sum of positive PDs diagnostic criteria, and the dimensions of the SAS  of the 91 patients who rated themselves on the SF-36 complied. The distribution of patients were cluster A+B group (n = 18), cluster C n = 21), and Axis I-disorder group (n = 11). Due to small sample sizes, the Axis I disorder was dropped from further analysis and the two PD groups were pooled as to the study of changes after treatment. The 39 PD patient with SF-36 ratings both at baseline and follow-up were compared to the 33 PD patients only seen at baseline. The diagnoses at follow-up are shown in Table 1, and AxFew significant differences were observed between compliant and non-compliant PD patients at follow-up Table . In partIn the 39 PD patients who complied at both baseline and follow-up, significant improvement was seen in the RF and MH dimensions of the SF-36, while considerable, but non-significant changes were observed for BP and SF Table .Both professional-rated measures the GAF, and the mean total number of PD criteria, showed significant improvement. Among the patient-rated measures significant better results at follow-up were found for the SCL-90-R PSI, the SAS overall adjustment, and the SAS social and leisure scales.The main findings of this study of mainly co-morbid PD patients treated at an ordinary POC, was that the QoL on both the physical and mental SF-36 dimensions was significantly lower than that of an age- and gender-adjusted general population sample. According to our knowledge, ours is the first report on QoL-data in such PD patients at a POC. This finding is in accordance with QoL studies of PD patients in the general population ,13, and In contrast to the epidemiological study from Australia  we did nComorbid Axis I disorders explained a significant part of scores of PF, GH, VT, SF, and MH scores of the total PD group. This is in accordance with the findings of the Australian study .We found that the SF-36 dimensions had variable associations with established psychiatric measures. As expected the SF-36 MH was most strongly associated with the psychiatric measures, but so were also SF and VT. For the SAS we found that overall adjustment and social and leisure activities were significantly correlated to all the SF-36 dimensions. In our PD sample we observed a somewhat different pattern of significant correlations between the GAF and the SF-36 dimensions than reported by Meijer et al.  in patieFor both the patient- and professional-rated psychiatric measures significant changes at follow-up after treatment was observed in the 39 patients who also scored themselves on the SF-36. We cannot say if these changes were related to treatment, and ours is not an outcome study. We wanted to examine if changes in established psychiatric measures were associated with changes in the QoL measured by the SF-36 in the PD patients seen at a POC.Significant changes at follow-up were found for only two of the SF-36 dimensions, however, one physical (RP) and one mental (MH). While the finding for MH was expected, the change in RP which covered problems with work or other daily activities as a result of physical health was more difficult to explain. The score on that dimension was extraordinarily low at baseline (mean 31.4), and regression towards mean could be a likely explanation. It seemed that only MH of the SF-36 changed in the same way as established psychiatric measures in our study. The SF-36 MH correlated significantly with most of such psychiatric measures, and MH is currently used as a valid measure for mental health in several studies [33].This result could indicate that the other dimensions of the SF-36 are less valid as measures of changes in mental health of PD patients, or alternatively that most aspects of QoL measured by the SF-36 do not change in PD patients even if established psychiatric measures do.The main strength of our study was that we were able examine systematically various aspects of the QoL measured by the SF-36 in a clinically relevant sample of PD patients at a POC which is a common setting for such patients in psychiatry.Our study had a number of weaknesses. The study groups were small with limited statistical power, and there was a considerable risk of type II errors. More significant differences as to the SF-36 dimensions could turn up in larger samples. Although we put considerable efforts into location of patients, we had a lower follow-up rate than we had expected. However, the PD patients who did not show up at follow-up did not differ much from those who did. We cannot, therefore, generalize the discrepancy observed between significant changes in established psychiatric measures and lack of such changes in most of the SF-36 dimensions of PD patients treated at a POC to widely.The same experienced psychiatrists did all the interviews at baseline and follow-up. Although she was not involved in any treatments, we cannot exclude an expectation bias from her side.We think that our study has to be considered an exploratory one. Our finding of a generally strongly reduced QoL should be replicated in a PD sample with less comorbid Axis I disorders, although their influence was limited. The same is true for QoL as a valid measure for change in PD patient, since it was not recommended as part of a standard outcome battery and was not used by major treatment studies of PD patients. However, our study confirmed that the SF-36 MH dimension seemed to be a valid psychiatric measure in our PD patient sample.In this study of the QoL in PD patients seen at an ordinary POC, we found that the PD patients had significantly lower mean scores on all the SF-36 dimensions compared to age- and gender-adjusted norm data. This is in accordance with the SF-36 measurements of other major diagnostic groups of mental disorders. Although the SF-36 dimensions correlated considerably with established psychiatric measures in our PD patients, they did not show the same significant changes over time as the established measures. The use of QoL measures like the SF-36 as an outcome measure in PD patients is in need of further investigation.BP: SF-36 Bodily painGAF: The Global Assessment of FunctioningGH: SF-36 General healthMH: SF-36 Mental healthPD: Personality disorderPDs: Personality disordersPF: SF-36 Physical functioningPOC: Psychiatric outpatient clinicPSI: Personality severity index of SCL-90-RQOL: Health-related quality of lifeRE: SF-36 Emotional role functioningRP: SF-36 Physical roleSAS: The Social Adjustment ScaleSCL-90-R: The Symptom Checklist 90-RevisedSF-36: MOS Short Form 36SF: SF-36 Social functioningVT: SF-36 VitalityThe author(s) declare that the have no competing interests.KN conceived and planned the study, prepared the therapists at Furuset Outpatient Department, did all the psychiatric interviews at baseline and follow-up, and drafted the manuscript. AM helped designing of the study, supervised the statistic calculations, and drafted the manuscript. AAD participated in the design and coordination of the study, and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Escherichia coli a model organism provides information for annotation of other genomes. Our analysis of its genome has shown that proteins encoded by fused genes need special attention. Such composite (multimodular) proteins consist of two or more components (modules) encoding distinct functions. Multimodular proteins have been found to complicate both annotation and generation of sequence similar groups. Previous work overstated the number of multimodular proteins in E. coli. This work corrects the identification of modules by including sequence information from proteins in 50 sequenced microbial genomes.E. coli K-12 proteins were identified from sequence similarities between their component modules and non-fused proteins in 50 genomes and from the literature. We found 109 multimodular proteins in E. coli containing either two or three modules. Most modules had standalone sequence relatives in other genomes. The separated modules together with all the single (un-fused) proteins constitute the sum of all unimodular proteins of E. coli. Pairwise sequence relationships among all E. coli unimodular proteins generated 490 sequence similar, paralogous groups. Groups ranged in size from 92 to 2 members and had varying degrees of relatedness among their members. Some E. coli enzyme groups were compared to homologs in other bacterial genomes.Multimodular E. coli paralogous groups correlates with known physiological and taxonomic relationships between the organisms.The deleterious effects of multimodular proteins on annotation and on the formation of groups of paralogs are emphasized. To improve annotation results, all multimodular proteins in an organism should be detected and when known each function should be connected with its location in the sequence of the protein. When transferring functions by sequence similarity, alignment locations must be noted, particularly when alignments cover only part of the sequences, in order to enable transfer of the correct function. Separating multimodular proteins into module units makes it possible to generate protein groups related by both sequence and function, avoiding mixing of unrelated sequences. Organisms differ in sizes of groups of sequence-related proteins. A sample comparison of orthologs to selected  Eschericia coli remains a useful resource to the genomic community as it provides important knowledge which can be applied to the analysis of most microbial genomes. Its central role devolves from two facts; first, the accumulated results of seven decades of laboratory experimentation have identified the function(s) of over half of its gene products; second being a metabolic generalist, E. coli's metabolic functions are widely shared among other organisms.E. coli's genes. While searches for sequence matches to unknown genes usually yield a large number of matches, chances are high that firm functional information comes only from experimental studies on E. coli. Because annotations of genes do not always indicate that the assignments are derived, and because derived annotations are used serially for further annotation without experimental confirmation, many genes carry original E. coli annotations. It is therefore important to the entire genome-analyzing community that the data on E. coli gene products be as accurate as possible. Since the original GenBank deposit of E. coli K-12 (U00096), new and updated annotations are available at NCBI (U00096.2) and at more specialized databases including, ASAP [E. coli annotations [Common practices of annotation rely, more than one might realize, on the accuracy of the annotation of ng, ASAP , EchoBASng, ASAP , EcoCyc ng, ASAP , GenProt-12 U0009, new andng, ASAP . An effootations .E. coli genome has led us to an appreciation of the pernicious role that gene fusions often play as troublemakers in function assignments and in relating groups of sequence similar proteins [Over recent years, our work on the proteins . The fusproteins  and Supeproteins  containsproteins .Annotation involving transfer of function from composite proteins to sequence similar matches requires that the alignment regions be evaluated in order to determine whether all activities or only one of them should be assigned to the matching sequence. Currently fused proteins are themselves not always annotated to reflect that they encode more than one function, and rarely is the location of the separate functions indicated. Different combinations of fused genes are seen in the sequenced genomes, adding potential sources for annotation errors. Errors in functional assignments including those caused by fused genes have been noted years ago  and thatE. coli proteins from partial alignments between proteins encoded in the E. coli genome [E. coli to detect proteins which are fused in the E. coli genome and are present as individual components in one of the other genomes. We have also made use of published experimental data on E. coli gene fusions. As a result the number of fused proteins in E. coli has been reduced to 109. The number of groups of sequence related proteins was also reduced from 609 to 490 since some of the previously identified groups are made up of protein domains catalyzing only part of an overall reaction. This work represents a revision of the state of fused proteins in the E. coli genome their affect on genome analysis both within E. coli and across genomes.Previous work has been published where we identified fused i genome . This woE. coli, they differ in sequence and activity. Distinctly different, multifunctional proteins are defined as those that carry out more than one reaction or activity in the same part of the protein. Examples of such multifunctional proteins are encoded by the genes cobU, birA, ubiG, folD, cysG, tesA, and ndk (for details see gene products at GenProtEC [To prevent confusion, we define multimodular proteins as those seeming to result from gene fusion in which two independent proteins are connected. Multimodular proteins encode separate functions in different parts of the molecule. These functions might be the same if two like elements have fused, or as we see more often in enProtEC ).E. coli [A protein that illustrates both properties is the FadB protein of E. coli ,18. FadBE. coli was completed, we saw that sequence similarity among its proteins was widespread [E. coli proteins as being multimodular and encoded by fused genes [E. coli proteins. However, we have since found that many of these so-called multimodular proteins were proteins containing more than one domain and not more than one protein. Such multidomain proteins may appear to encode two functions but in reality encode two or more conserved motifs . By including sequence information from other genomes besides E. coli we were able to better distinguish fusions of complete proteins versus the more common fusions of protein domains. Of the 287 proteins previously identified as multimodulars only 70 remained as fused proteins in this study with the remaining representing domain fusions.In earlier work, before the genomic sequence of despread ,19. Afteed genes ,20. The E. coli proteins long known to contain multiple functions encoded at separate parts of the proteins include GlnE [E. coli proteins and proteins encoded in 50 sequenced genomes. The component proteins of a multimodular protein may be unimodular and unfused in another genome. We looked for alignments between the larger potentially multimodular proteins in E. coli and smaller orthologous proteins that are homologous to only one of the modules . A list of the major types of multimodular proteins is shown in Table Table • Many of the multimodular enzymes function in the biosynthesis or degradation of compounds (amino acids, cofactors, peptidoglycan and fatty acids).• The majority of the multimodular transport proteins encode fusions of components of the ABC superfamily transporters (ATP-binding and membrane component). Also, fusions of the PTS proteins were detected in different combinations. Thirteen proteins contained two or more PTS components, including Hpr, enzymes I, IIA, IIB, or IIC.• Among the multimodular regulatory proteins, two-thirds were part of two-component regulatory systems and contained histidine kinases fused to response regulators. Seldom were known domain subdivisions within these modules detected by the rules we applied.While the fraction of enzymes (39%) is similar to the fraction of enzymes encoded in the genome as a whole (36%), the proportion of multimodular transport proteins (38%) and regulatory proteins (17%) were higher than their proportion genome wide (14% and 8% respectively). The over-representation in transporters and regulators is a reflection of the level of gene duplication seen for these proteins. Large paralogous groups are detected for some of the ABC transporter protein subunits and for components of the two-component regulators.E. coli proteins to each other using the AllAllDb algorithm of the Darwin package, we collected all aligned pairs with a similarity score of less than or equal to 200 PAM units, with an alignment of at least 83 residues. Altogether 9,626 unique pairs met these criteria .We used the data on pairwise similarity to assemble groups of proteins of similar sequence that were unlike other proteins in the cell. Besides the PAM less than 200 and alignment length of at least 83 residues, two additional requirements were imposed; that more than 45% of each protein in each pair be aligned, and that a module could not belong to more than one group. A transitive clustering process was used to form the sequence-similar groups . This grE. coli ' [see E. coli groups and their members]'. Altogether 1946 unimodular proteins belonged to one of the groups. Modules from 94 of the multimodular proteins were present in 61 of the groups. Table We identified 490 sequence-similar or paralogous groups in E. coli with known or predicted function, they tend to be present in smaller paralogous groups as compared to the transporters and regulators. Among the larger groups the oxidoreductases and the subunits of oxidoreductases are most common, making up 8 of the top 20 enzyme groups -binding oxidoreductases of E. coli. The number of homologous sugar kinases, respiratory reductase subunits, and nucleoside diphosphate (Nudix) hydrolases appeared overall to be lower in B. subtilis.In addition to using paralogous groups for intra-genomic analyses, the groups were also used in cross genome comparisons , while in Xylella fastidiosa, an aspartokinase is fused to a diaminopimelate decarboxylase (gi9106073). One needs to be alert to partial alignments. In this case, the annotation is correct for both activities of the Xylella protein, although the description does not follow the convention of stating the N-terminal activity first, raising the potential for misidentification of the activity of a partial homolog.As shown in Figure E. coli and X. fastidiosa connected proteins acting in the same pathway. However, the pathways are different for the two organisms, threonine biosynthesis for E. coli and lysine biosynthesis in X. fastidiosa. Fusions of genes in a pathway have long been known and also the fusions of different genes in different organisms. In the tryptophan biosynthesis pathway of E. coli both the trpC gene (formerly trpC(F)) and the trpD gene (formerly trpG(D)) encode two enzymes as indicated in their former names. In contrast Rhizobium meliloti has a fusion between the trpE and trpG genes, trpE(G) [The details of gene duplication and divergence and of gene fusions have followed different courses in separate lines of descent of bacteria. The fusions of different gene partners to aspartokinase in  trpE(G) . Such diTo promote awareness of fused proteins, databases should list such proteins with their separate component activities and the approximate locations of these; either by start and end residues, or by module location . Such a format has been implemented in GenProtEC . When anE. coli proteins similar in sequence vary in size from two (simple pairs) up to 92 members -binding oxidoreductases is much larger in B. subtilis (31 proteins) than in the enterics (18 proteins). The B. subtilis enzymes in this group are fatty acid biosynthesis enzymes, agreeing with the known fact that this organism synthesizes a greater variety of fatty acids and has dedicated more of its proteome towards diversifying its fatty acid biosynthetic capabilities [In another example, bilities ,34. Thus• Proteins encoded by fused genes, multimodular proteins, require special attention in genome analysis. Such multimodular proteins contain two or more functional components that are located at separate parts of the protein and that may exist as independent proteins in other genomes. Annotation of the multimodular proteins should include the separate functions and their corresponding locations in the gene product. This will improve transfer of function between the fused proteins and sequences matching their entire length or only the length of one of their module components. Current annotation errors involving fused genes can be remedied by introducing this approach.E. coli was improved by making use of sequence information from 50 genomes to detect alignments between the fused proteins and smaller, un-fused homologs corresponding to the component modules. The more common multidomain proteins, proteins containing fused sequence domains or motifs that together make up one overall function, were not detected as multimodular proteins by this approach. As a result the current number of fused E. coli proteins was reduced to 109 proteins with 8 containing three modules and 101 containing two modules. The multimodular E. coli proteins consist mainly of enzymes, regulators and transport proteins. Their component modules are often not related by sequence but many are related in that they function in a common pathway or cell role. Components of fused genes appear to vary from genome to genome hence complicating their detection and function assignment.• The identification of multimodular proteins in • Multimodular proteins are different from multifunctional proteins in that the latter catalyze more than one reaction in the same region of the protein.E. coli proteins ranging in size from 2 to 92 were generated from the new module data. The smaller groups range widely in degree of relatedness while the larger groups have diverged from one another to about the same extent. Transport proteins and regulatory proteins were found in the larger groups while enzyme groups tended to have fewer members.• The generation of paralogous or sequence related groups is improved when the modules of multimodular proteins are separated and treated as independent proteins for the grouping process. 490 groups of sequence related E. coli proteins belong to paralogous groups, reflecting the prominent role of duplication and divergence in the evolution of the genome. The number and sizes of paralogous groups reflect the distinctiveness of the organisms and they can be used in cross genome comparisons.• Over half of the Aquifex aeolicus, (AE000657); Archaeoglobus fulgidus, (AE000782); Aeropyrum pernix, (BA000002); Agrobacterium tumefaciens, (AE007869/AE007870); Borrelia burgdorferi, (AE000783); Bacillus halodurans, (BA000004); Bacillus subtilis, (AL009126); Buchnera sp. APS, (BA000003); Campylobacter jejuni, (AL111168); Clostridium acetobutylicum, (AE001437); Chlamydia muridarum, (AE002160); Chlamydophila pneumoniae CWL029, (AE001363); Deinococcus radiodurans, (AE000513/AE001823); Escherichia coli K-12, (U00096); Escherichia coli O157:H7 EDL933, (AE005174); Escherichia coli O157:H7, (BA000007); Haemophilus influenzae, (L42023); Helicobacter pylori 26695, (AE000511); Halobacterium sp. NRC-1, (AE004437); Lactococcus lactis subsp.lactis, (AE005176); Mycobacterium leprae, (AL450380); Mycoplasma genitalium, (L43967); Mycobacterium tuberculosis H37Rv, (AL123456); Methanococcus jannaschii, (LL77117); Mesorhizobium loti, (BA000012); Mycoplasma pneumoniae, (U00089); Mycoplasma pulmonis, (AL445566); Methanobacterium thermoautotrophicum, (AE000666); Neisseria meningitidis MC58, (AE002098); Pseudomonas aeruginosa, (AE004091); Pyrococcus horikoshii, (BA000001); Pasteurella multocida, (AE004439); Pyrococcus abyssi, (AL096836); Rickettsia prowazekii, (AJ235269); Salmonella enterica subsp. enterica serovar Typhi, (NC_003198); Salmonella typhimurium LT2, (AE006468); Shewanella oneidensis MR-1, (NC004347); Sinorhizobium meliloti, (AL591688); Staphylococcus aureus subsp.aureus Mu50, (BA000017); Streptococcus pneumoniae TIGR4, (AE005672); Streptococcus pyogenes M1 GAS, (AE004092); Sulfolobus solfataricus, (AE006641); Synechocystis PCC6803, (AB001339); Thermoplasma volcanium, (BA000011); Thermotoga maritima, (AE000512); Treponema pallidum, (AE000520); Ureaplasma urealyticum, (AF222894); Vibrio cholerae, (AE003852/EC003853); Xylella fastidiosa 9a5c, (AE003849); Yersinia pestis, (AL590842).Protein coding sequences were obtained from GenBank and included the following genomes: Pairwise sequence alignments and scores were generated using the AllAllDb program of Darwin , version 2.0, developed at the ETHZ in Zurich . MaximumFor the work reported here, sequence pairs were collected that had alignment lengths of at least 83 amino acids and distances of 200 PAM units or less. We chose the length requirement of 83 residues as it improves the significance of the sequence alignments for the more distantly related protein pairs . The reqE. coli literature and from unequal sequence alignments. The literature was searched for E. coli proteins with more than one function encoded at separate parts of the protein. The locations of the alignment regions in the proteins were analyzed for orthologous and paralogous protein pairs. We identified proteins with two or more non-overlapping alignment regions where each region aligned separately to smaller homologs. Figure Proteins encoded by fused genes were identified from the E. coli were aligned against themselves. Protein pairs aligning with >45% of the length of the peptides were used in a transitive grouping process as previously described [The sum of the separated modules from the multimodular proteins and the naturally occurring unimodular proteins of escribed . The traMS designed the study, performed the sequence analysis, and participated in the data analysis and in writing the manuscript. MR participated in the data analysis and in writing the manuscript.E. coli proteins. The table contains a complete list of the multimodular proteins in E. coli. Each module is described by its Gene name, Module Id, Module Start and End positions, Gene type, and Module Product.Multimodular Click here for fileE. coli. The members of the 409 paralogous groups are indicated by their Group Membership, Module Id, Module Start and End Position, Module Product.E. coli paralogous groups and their members. The table contains a complete list of the paralogous protein groups in Click here for file


Acute myocardial infarction (MI) following anaphylaxis is rare, especially in subjects with normal coronary arteries. The exact pathogenetic mechanism of MI in anaphylaxis remains unclear.The case of a 32-year-old asthmatic male with systemic anaphylaxis, due to oral intake of 500 mg amoxycillin, complicated by acute ST-elevation MI is the subject of this report. Following admission to the local Health Center and almost simultaneously with the second dose of subcutaneous epinephrine (0.2 mg), the patient developed acute myocardial injury. Coronary arteriography, performed before discharge, showed no evidence of obstructive coronary artery disease. In vivo allergological evaluation disclosed strong sensitivity to amoxycillin and the minor  determinants of penicillin.Acute ST-elevation MI is a rare but potential complication of anaphylactic reactions, even in young adults with normal coronary arteries. Coronary artery spasm appears to be the main causative mechanism of MI in the setting of "cardiac anaphylaxis". However, on top of the vasoactive reaction, a thrombotic occlusion, induced by mast cell-derived mediators and facilitated by prolonged hypotension, cannot be excluded as a possible contributory factor. Acute myocardial infarction (MI) complicating anaphylaxis induced by drugs or other chemicals is uncommon and only sporadic cases have been reported -9. The uA 32-year-old man was admitted to the emergency room of the local Health Center because of anaphylaxis, which developed 2 hours and 15 minutes after the ingestion of amoxycillin (500 mg), prescribed by his dentist. Prodromal signs of anaphylaxis (flushing, pruritus, warmth, urticaria) reportedly occurred about 15 minutes before the onset of symptoms from other systems. Ten days earlier the patient, an asthmatic since childhood, had completed a 4-day course of amoxycillin (500 mg TID) without any side effects. Apart from obesity, there were no other risk factors for coronary artery disease.2) with generalized erythema, angioedema, cyanosis and diffuse wheezing; the systolic blood pressure was 70 mmHg and the pulse rate 120 bpm. The patient was then connected to a cardiac monitor, which showed sinus tachycardia (approximately 140 bpm) without ST-segment and T wave abnormalities  of 90%.On admission the patient was in acute distress. He was complaining of dizziness, blurred vision, dyspnea and abdominal pain. Initial examination revealed an obese man  was injected subcutaneously (SC); dimethindene (4 mg) and hydrocortizone (500 mg) were administered intravenously (IV) in slow infusion. Nebulized salbutamol and supplemental oxygen were given as well. Normal saline with 50 mg ranitidine hydrochloride and Ringer's solution were infused through separate intravenous lines.3 to V6  a second dose of epinephrine 0.2 mg) was given SC 20 minutes later. Almost simultaneously with the administration of the second dose of epinephrine, ST-segment elevation appeared on the monitor Fig.  and the  mg was gth hospital day showed preserved systolic function (ejection fraction 60%) without wall motion abnormalities. Left  were performed using penicilloyl polylysine (PPL), and minor determinant mixute (MDM) [supplied as Allergopen by Allergopharma (Reinbeck, Germany)]; amoxycillin, ampicillin and cefamandole solutions were also used (concentration 20 mg/ml) for PST. PST were strongly positive [4+ reaction  i.e. wheal >5 mm in diameter with pseudopodes, with no reaction at all to the diluent control] to amoxycillin and ampicillin. Intradermal tests were performed only to PPL, MDM (dilution 1/10) and cefamandole (0.2 mg/ml and 2 mg/ml) and resulted in strongly positive reaction (4+) only to the MDM. Circulating specific IgE to penicillin V, penicillin G, amoxycillin, ampicillin and cefaclor was not demonstrable by CAP (Pharmacia, Sweden).Anaphylactic reactions may trigger cardiovascular events, including MI -9, primaAcute coronary syndromes following antibiotic-induced anaphylaxis is uncommon and only few cases have been reported so far ,4,12-14.Evidence that the human heart is the target as well as the site of anaphylaxis is constantly growing. A number of studies by Marone et al ,16 and oIn the present case, the temporal sequence of events suggests that cardiac anaphylaxis was the triggering factor of MI. We propose that prolonged coronary vasospasm induced by vasoactive and inflammatory mediators, released during anaphylaxis, was the main causative mechanism. However, in view of the persistence of chest pain following IV nitroglycerin infusion, and its complete resolution promptly after IV thrombolysis, a thrombotic vascular occlusion, on top of the vasospastic reaction, cannot be excluded. The latter is also supported by the prolonged systemic hypotension, which, as it has been emphasized in previously reported cases , probablThe administration of epinephrine – a life saving agent in cases of anaphylaxis – has been implicated as a cause of acute MI in a limited number of reports ,28. In tThe allergological evaluation performed in our patient showed strong sensitivity to amoxycillin and to the minor determinants, which are the allergenic epitopes associated with systemic anaphylaxis. The negative CAP results do not negate the in vivo findings, since it is well established that the in vitro techniques are not as sensitive as skin tests. Furthermore, CAP detects antibodies against the major determinants of penicillins  and not to the minor ones, which are implicated in systemic anaphylaxis. For technical reasons, relating to both the Allergology Center and the patient's professional obligations as well as residence distance, the allergological work up was completed 6 months later; such a delay might also have contributed to the negative in vitro findings.Acute ST-elevation MI is a rare but potential complication of anaphylactic reactions, even in young adults with normal coronary arteries. Physicians should be alert for such a complication in order to diagnose it early and treat properly.It is fairly well established that that human heart can be both, the site and the target of severe anaphylaxis; in this setting cardiac mast cells – activated and releasing multiple vasoactive mediators – play an important role in pathogenesis of cardiac complications -18.In the above case, mediator-induced coronary artery spasm was the main, but probably not the exclusive causative mechanism of anaphylaxis-related MI. The thrombotic vascular occlusion, induced by inflammatory mediators and facilitated by prolonged hypotension, cannot be excluded as a possible contributory factor.ECG = electrocardiogram; IV = intravenously; MDM = minor determinant mixute; MI = myocardial infarction; NV = normal values; PPL = penicilloyl polylysine; PST = prick skin tests; SC = subcutaneously.The author(s) declare that they have no competing interests.AG was responsible for the initial evaluation and management of the patient, GL was involved in the cardiological evaluation and management and, KK-F performed the allergological evaluation. All authors have equally contributed in the preparation and revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm) assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF) structures when a green fluorescent protein (GFP) “head” is added to the N terminus. The localization of this GFP-AD-Cterm to the cleavage furrow of dividing Dictyostelium cells depends on assembly state, similar to full-length myosin-II. This tail fragment therefore represents a good model system for the regulated formation and localization of BTFs. By reducing regulated BTF assembly to a more manageable model system, we were able to explore determinants of myosin-II self-assembly. Our data support a model in which a globular head limits the size of a BTF, and the large-scale charge character of the AD-Cterm region is important for BTF formation. Truncation analysis of AD-Cterm tail fragments shows that assembly is delicately balanced, resulting in assembled myosin-II molecules that are poised to disassemble due to the phosphorylation of only three threonines.Myosin-II thick filament formation in  A portion of the myosin tail coupled to a green fluorescent protein "head" proves a valuable model for understanding myosin self-assembly Myosin-II (hereafter referred to as myosin) is a hexameric protein composed of two heavy chains, two regulatory light chains, and two essential light chains A. The heDictyostelium discoideum assembles without the aid of a cofactor, demonstrating that the myosin molecule itself contains all the information needed to form a highly organized structure of a defined size (Dictyostelium (A bipolar thick filament (BTF) is a highly organized structure composed of individual myosin molecules. In vitro, myosin from the cellular slime mold ned size . While postelium .Dictyostelium, phosphorylation of myosin inhibits filament assembly  heavy chain of myosin is sufficient to inhibit BTF formation . These t in vivo . Mutatio in vivo . The dou in vivo .Dictyostelium myosin tail occurs every 196 a.a.  produces a chimera that is apparently indistinguishable from full-length myosin in its ability to form BTFs  and 3xAla can localize, have demonstrated the necessity of BTF assembly for proper localization of myosin . Howeverum cells . Chimerisin tail . This hasin tail .Dictyostelium BTFs has identified functional domains and repeating patterns in the coiled-coil tail. How do these properties come together to form a BTF that can be regulated by phosphorylation of only three a.a.? How do these properties contribute to regulated assembly? How do these properties enable a myosin BTF to localize to the cleavage furrow during cytokinesis? We have approached these questions by reconstituting regulated assembly of BTFs and defining the minimal part of the molecule required for regulated BTF assembly in vitro and in vivo.Characterization of We wished to test whether both the Ala 1 and the Ala 2 domains of myosin are necessary to regulate BTF assembly  or whethThick filaments and paracrystals efficiently sediment upon centrifugation, whereas unassembled molecules remain soluble. Wild-type full-length myosin efficiently assembles in buffers of intermediate ionic strength (25–100 mM NaCl), while phosphorylated full-length myosin is unassembled A Kuczma. Therefo222) (unpublished data). Therefore, both tail fragments behave as two-stranded coiled-coils in these assays.We next tested whether AD-Cterm (3xThr) and AD-Cterm (3xAsp) tail fragments form coiled-coils, similar to full-length myosin. The oligomerization state of the tail fragments was determined by sedimentation equilibrium analysis, and the secondary structure was assessed by circular dichroism (CD). Neither tail fragment is expected to assemble in the buffers used for these studies, as they contain 500 mM NaCl. Sedimentation runs were carried out as a function of protein concentration, and equilibrium traces were obtained after 20 h of centrifugation. The profiles were fit to an equation describing the sedimentation behavior of a single, non-associating, ideal species B. The diThe sedimentation equilibrium analysis demonstrates that a direct comparison of coiled-coil stability can be made by thermal denaturation at 500 mM NaCl because neither tail fragment self-assembles to form larger species in this condition. Thermal melts are reversible and at equilibrium and far-UV CD spectra show both tail fragments are α-helical at the starting temperature of the melt (4 °C) and are random coil at the ending temperature (60 °C) (unpublished data). The melting temperature for the AD-Cterm (3xThr) and AD-Cterm (3xAsp) tail fragments at both protein concentrations are identical , it forms paracrystals with a 14-nm (98-a.a.) periodicity, corresponding to the periodicity of charge in the AD-through-C-terminal region of the myosin tail (The AD-Cterm data show that tail fragment solubility accurately predicts the bulk solubility properties of full-length myosin. However, they do not form true BTFs. When AD-Cterm (3xThr) is assembled in buffer containing 50 mM NaCl with 10 mM MgClsin tail  . Both GFP-AD-Cterm (3xThr) and GFP-AD-Cterm (3xAsp) versions of this protein were made to mimic the unphosphorylated and phosphorylated states of myosin, respectively.To test whether the presence of a globular domain might induce AD-Cterm to form regulated, bipolar structures analogous to the thick filaments formed by full-length 2 and imaged them using EM. Bipolar structures first formed on a time scale of 2–5 min  apart, corresponding well to the 14.3-nm offset of full-length myosin heads in a BTF (We assembled purified GFP-AD-Cterm (3xThr) in buffer containing 50 mM NaCl with 10 mM MgCl 2–5 min B, then bhere see , it formhere see . The bipin a BTF .n = 63) at the center of the bare zone (area where heads are absent), close to the width of full-length myosin BTFs (33 ± 1 nm). For both GFP-AD-Cterm and full-length myosin, the length of the bare zone is approximately equal to the length of the coiled-coil. This length is 130–190 nm for full-length myosin and 85 ± 11 nm (n = 63) for GFP-AD-Cterm. This is an indication that GFP-AD-Cterm probably assembles in a manner similar to full-length myosin, with differences in BTF dimensions reflecting differences in myosin tail length.We compared the dimensions of GFP-AD-Cterm (3xThr) and full-length myosin BTFs . The widNotably, the solubility properties of GFP-AD-Cterm (3xThr) and GFP-AD-Cterm (3xAsp) C are simDictyostelium in vivo, we expressed our constructs in myosin-null Dictyostelium cells. However, the GFP-tail fragments were vastly overexpressed in vivo (unpublished data). Introducing the 31-a.a. regulatory light chain (RLC) binding site between the GFP and tail fragment sequences (Dictyostelium cells. To ensure that the RLC did not interfere with filament formation, we assayed for assembly of the GFP-RLC-tail fusions in Dictyostelium cell extracts. As expected, the 3xThr and 3xAla GFP-RLC-AD-Cterm proteins were assembly-competent in Dictyostelium extracts, while GFP-RLC-AD-Cterm (3xAsp) was not (unpublished data), demonstrating that the RLC does not interfere with regulated filament formation.To test the ability of GFP-tail fragments to localize to the cleavage furrow of equences  producedDictyostelium cells . Dictyostelium cell during cytokinesis. In these cells, GFP-myosin is recruited to the cell equator early during cytokinesis and remains there until the cell divides. Thereafter, myosin remains at the back end of the daughter cell as they move away from one another. In contrast, the labeled beads of similar size show no directed motion toward the furrow  and, interestingly, this GFP-AD (1xThr) tail fragment had bulk sedimentation properties very similar to those of headless AD (1xThr), suggesting that the presence of a globular domain does not affect in vitro solubility, similar to AD-Cterm and GFP-AD-Cterm tail fragments B. The ADTo determine whether the entire 34-kDa portion of the tail C-terminal to the AD is required for regulated assembly, we generated a series of tail fragments that all start at the AD and are truncated at different positions within the C-terminal 34 kDa of the tail see C. This iTo test whether adding on a larger portion of the tail results in a greater degree of regulated assembly, we constructed tail fragments that start at the AD and end at a.a. 1,966, the end of Ala 2 (AD-Ala 2). As with other tail fragments, the aspartic acid variant of this construct is more soluble than its wild-type counterpart D. SurpriWhen the entire C-terminal domain is included in the AD-Cterm tail fragment, the inhibitory effect of Ala 2 is overcome. To test whether this stabilizing effect is specific to the C-terminal domain, we determined if Ala 1 could also stabilize assembly. A tail fragment was constructed that starts at Ala 1 and ends at Ala 2 (Ala1-Ala2 [2xThr]). The salt-dependent assembly of Ala1-Ala2 (2xThr) is more efficient than that of AD-Ala2 (2xThr) E at 5 μMThe 196-a.a. repeat is remarkably symmetric in the entire AD-Cterm region A. We cren = 118) apart, consistent with the 14-nm spacing of full-length Dictyostelium myosin. The AD-Cterm-GFP (3xThr) structures have a shorter bare zone than GFP-AD-Cterm (3xThr) structures (62 ± 8 [n = 75] versus 85 nm), but a similar width to both GFP-AD-Cterm (3xThr) and full-length myosin BTFs (32 ± 5 nm [n = 159] versus 27 nm and 33 nm, respectively). While the AD-Cterm-GFP (3xThr) structures differ slightly from those formed by full-length myosin and GFP-AD-Cterm (3xThr), it is clear that the presence of the globular domain leads to a fixed length, even in C-terminally capped myosin tail fragments.To test whether AD-Cterm-GFP (3xThr) can form BTFs, we performed EM B and sedWe next tested whether AD-Cterm-GFP assembly is regulated. AD-Cterm-GFP (3xAsp) is soluble at all NaCl concentrations, whereas AD-Cterm-GFP (3xThr) sediments efficiently between 25 mM and 150 mM NaCl C, very sThe assembly properties of both AD-Cterm (3xThr) and AD-Cterm (3xAsp) closely parallel those of full-length unphosphorylated and phosphorylated myosin described by The fact that GFP-AD-Cterm (3xThr) forms BTFs whereas AD-Cterm (3xThr) does not, demonstrates that the mere presence of a globular head differentiates BTFs from paracrystals, and that neither the myosin head nor the S2 region of the myosin coiled-coil is critical for BTF formation  is less robust than full-length myosin, constitutively assembled GFP-RLC-AD-Cterm (3xAla) always localizes to the cleavage furrow in cytokinesis see . In contThe difference in localization efficiency between full-length myosin and GFP-RLC-AD-Cterm (3xThr) might be due to differences in degree of assembly. The critical concentration of full-length myosin is estimated to be less than 20 nM . One migDictyostelium cells  and AD-Cterm (3xAsp) are very similar to full-length, wild-type myosin and full-length, phosphorylated myosin, respectively, show that Ala 1 is not required for regulated assembly. This result has been confirmed in vivo by deleting Ala 1 from both full-length wild-type and 3xAsp myosin (W. Liang and JAS, unpublished data). The CD and analytical ultracentrifugation data show that AD-Cterm (3xAsp) has not simply failed to fold into a two-stranded α-helical coiled-coil. These data are consistent with rotary shadowed electron micrographs of full-length 3xAsp and phosphorylated myosin. Gross destabilization of the coiled-coil would result in a single-headed myosin, and none have been observed . Global Small numbers of assembled AD-Cterm (3xAsp) paracrystals were seen in electron micrographs of samples (unpublished data). These paracrystals, although quite rare, possess a 14-nm periodicity similar to the wild-type tail fragment. Consistent with this observation, approximately 10% of 3xAsp myosin sediments at 50 mM NaCl, demonstrating that regulation of assembly is not an all-or-none process. Together with the CD data, this result indicates that AD-Cterm (3xAsp) is structurally and functionally intact. Rather than being conformationally disrupted, the 3xAsp tail fragment fails to assemble because the critical concentration for assembly is higher than that of the wild-type tail fragment.The fact that AD-Cterm-GFP forms BTF-like structures with heads on the outside and tails in the center demonstrates the role of the 196-a.a. charge repeat in assembly. This charge repeat is symmetric in the AD-Cterm region of the tail see A. If theInterestingly, AD-Cterm-GFP (3xThr) structures are shorter than GFP-AD-Cterm (3xThr) filaments, and the bare zones are smaller as well. This may be because C-terminal sequence elements in the myosin tail may be required for proper formation of the bare zone and thereby determine the exact morphology of the BTF.The truncation analysis provides evidence of a delicate balance between forces that drive assembly and disassembly of myosin tails. This is most strikingly demonstrated by the observation that Ala 2 was actually found to inhibit the assembly of AD, while Ala 1 and the C-terminal domain help to drive assembly. We propose that that this delicate balance is related to the 196-a.a. charge repeat in the tail. The attachment of Ala 2 purple,  to the ABecause the charge repeats likely play a role in each step in the assembly pathway, a small effect, such as the introduction of negative charge at key points in the molecule, could have a large effect on overall self-assembly. The threonine phosphorylation sites are positioned near the positive clusters of charge at the end of AD and in the C-terminal domain, suggesting that regulation by phosphorylation might have its largest effect on this charge pattern.Dictyostelium expression vector (pBIG) containing either GFP (3xThr) myosin or GFP (3xAsp) myosin (The polymerase chain reaction (PCR) was used to amplify the regions of a ) myosin . An NdeIDictyostelium myosin: AD (1xThr and 1xAsp), a.a. 1,531–1,824; AD-Cterm (3xThr and 3xAsp), a.a. 1,531–2,116; extended AD (2xThr and 2xAsp), a.a. 1,531–1,840; AD-Ala 2 (2xThr and 2xAsp), a.a. 1,531–1,966; AD-2015 (2xThr), a.a. 1,531–2,015; Ala 1-Ala 2 (2xThr), a.a. 1,348–1,966; Ala 1-Cterm (3xThr), a.a. 1,348–2,116  was used to eliminate the stop codon immediately before the SacI site in the AD-Cterm tail fragments contained in pET21a. The modified GFP-UV was subcloned into this vector using SacI and NotI. All DNA sequences were verified.Dictyostelium, the myosin regulatory light chain binding site (RLCBS) sequence was ligated into plasmid pTX-GFP -RIL strain (Stratagene). LB media contained 34 μg/ml chloramphenicol and either 100 μg/ml kanamycin for pET28a or 100 μg/ml carbenicillin for pET21a. Cells were grown at 37 °C to an absorbance at 600 nm of approximately 0.6. Protein expression was induced by adding 1 mM IPTG and incubating for 1 h. Cells were harvested by centrifugation at 6,370 × g for 15 min.Harvested cells were resuspended in 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM DTT, 500 mM NaCl, 30% sucrose, and protease inhibitor (PI) cocktail . For a single protein prep, the pellet from 10 l of culture was resuspended in buffer for a final volume of 40 ml. Cells were added dropwise into liquid nitrogen and stored at –80 °C.2 were added. The cells were lysed with two passes through a French Press (American Instrument Company, Silver Spring, Maryland, United States) at 10,000 pounds per square inch. After the first pass, a new batch of the PI cocktail was added. The lysate was centrifuged at 100,000 × g for 30 min at 4 °C. The supernatant was boiled for 10 min, and then a new batch of the PI cocktail and 1 mM DTT was added. The boiled supernatant was centrifuged at 100,000 × g for 30 min at 4 °C and then dialyzed against DEAE Low-Salt Buffer . The protein was injected onto a column consisting of eight tandem HiTrap DEAE Sepharose Fast Flow columns . The column was washed with 5 volumes of DEAE-LSB and protein was eluted on a linear gradient from 0 to 500 mM NaCl over 10 volumes. Fractions containing protein  were identified by SDS-PAGE. Protein was precipitated by addition of 85% (NH4)2SO4, stirred at 4 °C for 30 min, and centrifuged at 100,000 × g for 30 min at 4 °C. The pellet was resuspended in a minimal volume of gel filtration buffer (10 mM Tris [pH 8.0], 1 mM EDTA, 1 mM DTT, and 500 mM NaCl) and loaded on a HiLoad 26/60 Superdex 200 column (Amersham Biosciences). Fractions were analyzed by SDS-PAGE, and those containing pure tail fragments were pooled and dialyzed against Mono-Q Low Salt Buffer . Protein was injected onto a Mono Q HR 5/5 column (Amersham Biosciences) and a gradient from 0 to 1 M NaCl was run. Tail fragments typically eluted at 450 mM NaCl.Cells were thawed and 5 μg/ml RNase A , 50 μg/ml RNase-free DNase I , and 10 mM MgClProtein concentration was determined by measuring the absorbance at 280 nm in 6M guanidine hydrochloride. The extinction coefficient was calculated using the sequence of one strand of the coiled-coil as described in After harvesting, cells were resuspended in 50 mM sodium phosphate (pH 8.0) with 250 mM NaCl and containing PI cocktail. For a single protein prep, the pellet from 30 l of culture was resuspended in buffer for a final volume of 120 ml.2 was added along with the DNase I and RNase A. The supernatant was batch-bound to Ni-NTA resin  at 4 °C for 1 h. The resin was washed with 40 ml of 50 mM sodium phosphate (pH 8.0) with 250 mM NaCl (starting buffer) followed by 20 ml of starting buffer containing 12.5 mM imidazole. Protein was eluted from the column using approximately 5 ml of starting buffer with 500 mM imidazole. Fractions containing protein were loaded onto a Hi-Load 26/60 Superdex 200 prep grade gel filtration column (Amersham). Both GFP-AD-Cterm and AD-Cterm-GFP tail fragments elute between 120 ml and 150 ml. GFP-AD elutes between 152 ml and 174 ml. Protein was dialyzed against Mono-Q LSB overnight and then injected onto the Mono-Q HR 5/5 column (Amersham Biosciences) as described above. Protein elutes at about 450 mM NaCl. Protein was used immediately after purification for microscopy and sedimentation assays.Lysis and the first centrifugation step were identical to the myosin tail fragment protocol described above except that 5 mM MgCl2 concentration of 10 mM, and a final NaCl concentration of 50 mM. This solution was deposited onto glow-discharged 300-mesh carbon stabilized copper grids coated with formvar . AD-Cterm and AD proteins were assembled for 2 h, while GFP-containing proteins were assembled for 2 min. A 1% uranyl acetate solution was applied before imaging on a JEOL 1230 transmission electron microscope (JEOL USA, Peabody, Massachusetts, United States).Purified GFP-tail fragments were diluted to a final protein concentration of approximately 1 μM, a final MgClPurified protein was dialyzed overnight against 10 mM imidazole (pH = 7.5), 0.1 mM EDTA, and 1 mM DTT. 10 μM protein was added to an equal volume of 10 mM imidazole, 0.1 mM EDTA, 1 mM DTT, 2× mM NaCl. The final concentration of NaCl was × mM NaCl, where × = 0, 25, 50, 75, 100, 150, and 250 mM. Samples were incubated on ice for at least 30 min and then centrifuged at 132,000 × g for 15 min at 4 °C. Three replicates of each sample were made to ensure reproducibility. Samples of the supernatant and pellet were run on SDS-PAGE gels. The intensity of bands was quantified with an AlphaImager 2000 Documentation and Analysis System .222 was monitored with a 1 nm bandwidth. The temperature was increased in 1 °C increments, and the sample was equilibrated for 2 min at the new temperature before data collection. An averaging time of 30 s was used. The pmt dc voltage used was 1.0 V for 2.5 μM protein and 0.55 V for 50 μM protein. Kaleidagraph (Synergy Software, Essex Junction, Vermont, United States) was used for all data analysis. Plots of fraction denatured versus temperature were constructed as described in An Aviv Circular Dichroism Spectrometer model 62A DS was used (Aviv Corporation, Acton, Massachusetts, United States). The protein was in 10 mM Tris (pH 7.4), 500 mM NaCl, 1 mM EDTA, 1 mM DTT. Wavelength scans were taken from 260 nm to 200 nm. Data were collected every 1 nm with a 1-nm bandwidth and averaging time of 10 s. The spectra presented are buffer subtracted. Thermal melts were taken from 4 °C to 60 °C. θAD-Cterm (3xThr) was assembled by diluting the NaCl concentration to 50 mM. Assembled protein was recovered by centrifugation at 100,000 × g for 15 min. Pellets were resuspended in 10 mM Tris (pH 7.4), 0.1 mM EDTA, 500 mM NaCl  and centrifuged at 100,000 × g for 15 min to remove aggregates. The AD-Cterm (3xAsp) tail fragment was centrifuged at 100,000 × g for 15 min to remove aggregates. Both tail fragments were exchanged several times into High-Salt Buffer using an Amicon Ultra-15 30,000 Dalton cut-off ultra-filtration device (Millipore, Billerica, Massachusetts, United States). The High-Salt Buffer was the blank in all analytical ultracentrifugation experiments. After buffer exchange, the protein was centrifuged at 14,000 rpm for 10 min in a microcentrifuge to remove aggregates. The protein was snap-frozen in liquid nitrogen and shipped in dry ice to the Macromolecular Interactions Facility at UNC-Chapel Hill. The protein used for all analytical ultracentrifugation experiments was centrifuged at room temperature for 15 min at 16,000 × g to remove aggregates.All sedimentation equilibrium experiments were performed at the UNC Chapel Hill Macromolecular Interactions Facility as described in Dictyostelium cells (HS1 myosin-null um cells  were traum cells  supplemeDictyostelium cells expressing the appropriate construct was centrifuged and resuspended in 10 mM imidazole (pH 7.5), 0.1 mM EDTA, and 50 mM NaCl in the presence of protease inhibitors. The cells were lysed by freezing in liquid nitrogen and thawing. Cell lysates were allowed to sit on ice for 2 h before centrifugation at 132,000 × g for 20 min. The pellets were resuspended in equal volumes of the lysis buffer. The supernatant and pellets were resolved by SDS-PAGE without boiling the samples. The GFP-fusion proteins were imaged in the gel by excitation at 532 nm on a Typhoon 8000 imager .1 ml of nearly confluent Dictyostelium genome upstream of and in-frame with the gene encoding myosin-II (mhcA) by homologous recombination. The 167 bp just upstream of mhcA through the first 500 bp of the coding region were PCR-amplified from Dictyostelium genomic DNA using primers to add a 5′ XhoI site and a 3′ BamHI site. The PCR product was cloned into pBluescript digested with XhoI and BamHI. A PstI site was added between the upstream and mhcA coding regions by Quickchange mutagenesis (Stratagene) to create plasmid pBS/upstream-mhcA. GFP was amplified from pTX-GFP , a clonal cell line, GMO8B, was created by plating FACS-sorted GFP-positive cells on Klebsiella lawns and picking spores from individual GFP-positive plaques. The correct integration of GFP upstream of mhcA was verified by PCR and imaging of GFP fluorescence in-gel after SDS-PAGE of cell lysates. The cell line was able to develop normally and grow in suspension, and GFP localization in GMO8B was very similar to GFP-myosin localization in cells expressing GFP-myosin from a plasmid  in HL5 medium. The cells were immediately scraped off of the surface with a rubber policeman and transferred to a clean glass slide. After the cells were allowed to attach for approximately 30 min, the slide was rinsed in PBS to remove excess beads not taken up by the cells. The cells were then removed from the glass slide by pipetting up and down with HL-5 medium.Glass slides were coated with 10 μg/ml polylysine overnight. Approximately 1 ml of semi-confluent GMO8B Live cells were transferred to imaging chambers  in HL-5 medium. GFP and FluoSphere fluorescence were imaged at room temperature using a Zeiss (Oberkochen, Germany) Axiovert 200 inverted epifluorescence microscope equipped with a 63× objective (N.A. 1.3). Cells were imaged at 20-s intervals. Images were collected using Metamorph  and analyzed with ImageJ (NIH) and Photoshop .