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The apolipoprotein E (ApOE) ε4 allele is associated with neuropathological buildup of amyloid in the brain, and with lower performance on some laboratory measures of memory in some populations. In two studies, we tested whether ApOE genotype affects memory for everyday activities. In Study 1, participants aged 20-79 years old (n = 188) watched movies of actors engaged in daily activities and completed memory tests for the activities in the movies. In Study 2, cognitively healthy and demented older adults (n = 97) watched and remembered similar movies, and also underwent structural MRI scanning. All participants provided saliva samples for genetic analysis. In both samples we found that, in older adults, ApOE ε4 carriers demonstrated worse everyday memory performance than did ε4 noncarriers. In Study 2, ApOE ε4 carriers had smaller medial temporal lobes (MTL) volumes, and MTL volume mediated the relationship between ApOE genotype and everyday memory performance. These everyday memory tasks measure genetically determined cognitive decline that can occur prior to a clinical diagnosis of dementia. Further, these tasks are easily administered and may be a useful clinical tool in identifying ε4 carriers who may be at risk for MTL atrophy and further cognitive decline that is a common characteristic of the earliest stages of Alzheimer's disease.

Objective Antidepressant side effects are a significant public health issue, associated with poor adherence, premature treatment discontinuation, and, rarely, significant harm. Older adults assume the largest and most serious burden of medication side effects. We investigated the association between antidepressant side effects and genetic variation in the serotonin system in anxious, older adults participating in a randomized, placebo-controlled trial of the selective serotonin reuptake inhibitor (SSRI) escitalopram. Methods Adults (N = 177) aged ≥ 60 years were randomized to active treatment or placebo for 12 weeks. Side effects were assessed using the Udvalg fur Kliniske Undersogelser side-effect rating scale. Genetic polymorphisms were putative functional variants in the promoters of the serotonin transporter and 1A and 2A receptors (5-HTTLPR [L/S + rs25531], HTR1A rs6295, HTR2A rs6311, respectively). Results Four significant drug–placebo side-effect differences were found: increased duration of sleep, dry mouth, diarrhea, and diminished sexual desire. Analyses using putative high- versus low-transcription genotype groupings revealed six pharmacogenetic effects: greater dry mouth and decreased sexual desire for the low- and high-expressing serotonin transporter genotypes, respectively, and greater diarrhea with the 1A receptor low-transcription genotype. Diminished sexual desire was experienced significantly more by high-expressing genotypes in the serotonin transporter, 1A, or 2A receptors. There was not a significant relationship between drug concentration and side effects nor a mean difference in drug concentration between low- and high-expressing genotypes. Conclusion Genetic variation in the serotonin system may predict who develops common SSRI side effects and why. More work is needed to further characterize this genetic modulation and to translate research findings into strategies useful for more personalized patient care.

Older adults are among the most vulnerable to adverse cognitive effects of psychotropic medications and, therefore, the personalization of psychotropic treatment based on adverse drug reactions in this demographic is of great importance. We examined changes on neuropsychological tests of attention attributable to selective serotonin reuptake inhibitor (SSRI) treatment in anxious older adults. We also examined whether variation in serotonin receptor genes was associated with reduced attentional performance with SSRIs. We examined change from pre- to post-treatment in two attention measures – digit span and coding – in 133 adults aged ⩾60 yr with generalized anxiety disorder in a 12-wk trial of escitalopram vs. placebo. We also examined attentional change in relation to genetic variability in four central serotonin receptors: the serotonin transporter and serotonin 1A, 2A and 1B receptors. Digit span scores were significantly lowered in patients receiving escitalopram relative to placebo, indicating reduced attentional performance attributable to the SSRI. Individuals with high-transcription variants in the receptors 5-HTR2A rs6311 and 5-HTR1B rs11568817 had greater reductions in attention with SSRI treatment compared to placebo. We conclude that SSRIs reduce attention in older adults, particularly in those with high-expression genetic variants at the serotonin 2A and 1B receptors. Analysing neuropsychological changes with SSRIs in relation to genetic variation in the serotonin system may be a useful strategy for detecting subgroups of older adults who are more susceptible to side-effects of SSRIs. These results, if confirmed, could lead to the personalization of SSRI use to reduce adverse neurocognitive effects.

Background Generalized anxiety disorder (GAD) is a common disorder in older adults, which has been linked to hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis in this age group. The authors examined whether treatment of GAD in older adults with a selective serotonin reuptake inhibitor (SSRI) corrects this HPA axis hyperactivity. Methods The authors examined adults aged 60 years and older with GAD in a 12-week randomized controlled trial comparing the SSRI escitalopram with placebo. The authors collected salivary cortisol at six daily time points for 2 consecutive days to assess peak and total (area under the curve) cortisol, both at baseline and posttreatment. Results Compared with placebo-treated patients, SSRI-treated patients had a significantly greater reduction in both peak and total cortisol. This reduction in cortisol was limited to patients with elevated (above the median) baseline cortisol, in whom SSRI-treated patients showed substantially greater reduction in cortisol than did placebo-treated patients. Reductions in cortisol were associated with improvements in anxiety. Additionally, genetic variability at the serotonin transporter promoter predicted cortisol changes. Conclusions SSRI treatment of GAD in older adults reduces HPA axis hyperactivity. Further research should determine whether these treatment-attributable changes are sustained and beneficial.

The Alzheimer Disease Genetics Consortium (ADGC) performed a genome-wide association study of late-onset Alzheimer disease using a three-stage design consisting of a discovery stage (stage 1) and two replication stages (stages 2 and 3). Both joint analysis and meta-analysis approaches were used. We obtained genome-wide significant results at MS4A4A (rs4938933; stages 1 and 2, meta-analysis P (P(M)) = 1.7 × 10(-9), joint analysis P (P(J)) = 1.7 × 10(-9); stages 1, 2 and 3, P(M) = 8.2 × 10(-12)), CD2AP (rs9349407; stages 1, 2 and 3, P(M) = 8.6 × 10(-9)), EPHA1 (rs11767557; stages 1, 2 and 3, P(M) = 6.0 × 10(-10)) and CD33 (rs3865444; stages 1, 2 and 3, P(M) = 1.6 × 10(-9)). We also replicated previous associations at CR1 (rs6701713; P(M) = 4.6 × 10(-10), P(J) = 5.2 × 10(-11)), CLU (rs1532278; P(M) = 8.3 × 10(-8), P(J) = 1.9 × 10(-8)), BIN1 (rs7561528; P(M) = 4.0 × 10(-14), P(J) = 5.2 × 10(-14)) and PICALM (rs561655; P(M) = 7.0 × 10(-11), P(J) = 1.0 × 10(-10)), but not at EXOC3L2, to late-onset Alzheimer's disease susceptibility.

We sought to identify new susceptibility loci for Alzheimer's disease through a staged association study (GERAD+) and by testing suggestive loci reported by the Alzheimer's Disease Genetic Consortium (ADGC) in a companion paper. We undertook a combined analysis of four genome-wide association datasets (stage 1) and identified ten newly associated variants with P ĝ‰Currency sign 1 × 10 -5. We tested these variants for association in an independent sample (stage 2). Three SNPs at two loci replicated and showed evidence for association in a further sample (stage 3). Meta-analyses of all data provided compelling evidence that ABCA7 (rs3764650, meta P = 4.5 × 10 -17; including ADGC data, meta P = 5.0 × 10 -21) and the MS4A gene cluster (rs610932, meta P = 1.8 × 10 -14; including ADGC data, meta P = 1.2 × 10 -16) are new Alzheimer's disease susceptibility loci. We also found independent evidence for association for three loci reported by the ADGC, which, when combined, showed genome-wide significance: CD2AP (GERAD+, P = 8.0 × 10 -4; including ADGC data, meta P = 8.6 × 10 -9), CD33 (GERAD+, P = 2.2 × 10 -4; including ADGC data, meta P = 1.6 × 10 -9) and EPHA1 (GERAD+, P = 3.4 × 10 -4; including ADGC data, meta P = 6.0 × 10 -10). © 2011 Nature America, Inc. All rights reserved.

Objective: Generalized anxiety disorder (GAD) is common in older adults and can be treated with selective serotonin reuptake inhibitors (SSRIs). Genetic variation in the serotonin transporter gene promoter region is posited to be associated with SSRI efficacy: 2 polymorphisms (5-HTTLPR S/L and rs25531 g/a) form a haplotype with the La combination having higher transcription activity than other haplotypes. We hypothesized that GAD patients with no La haplotypes (La - ) have lower SSRI treatment efficacy than those with 1 to 2 La haplotypes (La + ). Method: The study enrolled subjects aged 60 years or older with a principal diagnosis of GAD into a 12-week, randomized trial of escitalopram versus placebo. One hundred fifty subjects were genotyped for the serotonin transporter promoter region haplotype and were divided into La - and La + genotype groups; the primary analyses were done in European Americans only (n = 125; n = 59 with escitalopram and n = 66 with placebo). Results: Escitalopram had no efficacy in the La - group versus moderate efficacy in the La + group. This genetic moderation of SSRI efficacy was due to a higher placebo response in La - subjects, compared with La + subjects. Drug concentration did not affect the genetic results. Exploratory analyses suggest that La - subjects had greater variability of anxiety symptoms unrelated to treatment. Conclusions: The serotonin transporter promoter haplotype is associated with variability in SSRI efficacy for late-life GAD. The variability may result from a genetic effect on anxiety symptom variability unrelated to treatment, rather than a pharmacodynamic effect that has been previously assumed. Further research is needed to understand the pharmacogenetic mechanism of this haplotype.

Strong evidence of linkage to late-onset Alzheimer disease (LOAD) has been observed on chromosome 10, which implicates a wide region and at least one disease-susceptibility locus. Although significant associations with several biological candidate genes on chromosome 10 have been reported, these findings have not been consistently replicated, and they remain controversial. We performed a chromosome 10–specific association study with 1,412 gene-based single-nucleotide polymorphisms (SNPs), to identify susceptibility genes for developing LOAD. The scan included SNPs in 677 of 1,270 known or predicted genes; each gene contained one or more markers, about half (48%) of which represented putative functional mutations. In general, the initial testing was performed in a white case-control sample from the St. Louis area, with 419 LOAD cases and 377 age-matched controls. Markers that showed significant association in the exploratory analysis were followed up in several other white case-control sample sets to confirm the initial association. Of the 1,397 markers tested in the exploratory sample, 69 reached significance (P

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is an autosomal dominant condition clinically characterized by behavioral, cognitive, and motor disturbances. Until now, at least 13 different FTDP-17 families that show linkage to chromosome 17q21 have been described. To characterize the FTDP-17 candidate region, flanked by the markers D17S1789 and D17S1804, we constructed a physical map in P1 and PAC clones. A detailed transcript map was generated by positioning known genes and EST clusters to the physical map. In total, we investigated 150 STSs mapped to this region. In addition, novel transcripts were isolated by exon-trapping. We were able to localize 19 known genes and a number of ESTs to this chromosomal region. Furthermore, seven novel genes were identified for which we isolated the full-length sequence.

Alzheimer’s disease (AD) pathology is associated with two proteins, the microtubule-binding protein tau and the -amyloid-precursor protein (APP). When tau becomes hyperphosphorylated, it forms neuritic aggregates, called neurofibrillary tangles. APP is cleaved by several enzymes to generate A peptides, which are – depending on their length – more or less amyloidogenic and form senile plaques. Pin1, a peptidyl-propyl cis/trans-isomerase, seems to be involved in both pathologies. Pin1 may facilitate dephosphorylation of tau by PP2A phosphatase, while cellular overexpression of Pin1 causes a reduction in the amyloidogenic processing of APP, making this enzyme an interesting target for pharmaceutical intervention. The gene encoding Pin1 maps to 19p13.2, a region previously linked to late-onset Alzheimer’s disease (LOAD). Therefore, Pin1 is an excellent positional and functional candidate for LOAD. In this study, we investigated whether common single nucleotide polymorphisms (SNPs) in Pin1 can influence the risk for developing late-onset Alzheimer’s disease. No association was observed with any of six polymorphisms or their resulting haplotypes. A meta-analysis of two promoter SNPs, which combined the data from this study with two previous ones, did not show any association either suggesting that common SNPs in Pin1 do not increase the risk for LOAD. © 2007 Elsevier Ireland Ltd. All rights reserved.

Objective: Recent genome-wide association studies (GWAS) of late-onset Alzheimer disease (LOAD) identified 9 novel risk loci. Discovery of functional variants within genes at these loci is required to confirm their role in Alzheimer disease (AD). Single nucleotide polymorphisms that influence gene expression (eSNPs) constitute an important class of functional variants. We therefore investigated the influence of the novel LOAD risk loci on human brain gene expression. Methods: We measured gene expression levels in the cerebellum and temporal cortex of autopsied AD subjects and those with other brain pathologies (∼400 total subjects). To determine whether any of the novel LOAD risk variants are eSNPs, we tested their cis -association with expression of 6 nearby LOAD candidate genes detectable in human brain ( ABCA7, BIN1, CLU, MS4A4A, MS4A6A, PICALM ) and an additional 13 genes ±100 kb of these SNPs. To identify additional eSNPs that influence brain gene expression levels of the novel candidate LOAD genes, we identified SNPs ±100 kb of their location and tested for cis -associations. Results: CLU rs11136000 ( p = 7.81 × 10 −4 ) and MS4A4A rs2304933/rs2304935 ( p = 1.48 × 10 −4 –1.86 × 10 −4 ) significantly influence temporal cortex expression levels of these genes. The LOAD-protective CLU and risky MS4A4A locus alleles associate with higher brain levels of these genes. There are other cis -variants that significantly influence brain expression of CLU and ABCA7 ( p = 4.01 × 10 −5 –9.09 × 10 −9 ), some of which also associate with AD risk ( p = 2.64 × 10 −2 –6.25 × 10 −5 ). Conclusions: CLU and MS4A4A eSNPs may at least partly explain the LOAD risk association at these loci. CLU and ABCA7 may harbor additional strong eSNPs. These results have implications in the search for functional variants at the novel LOAD risk loci.

Rare mutations in the gene encoding for tau (MAPT, microtubule-associated protein tau) cause frontotemporal dementia-spectrum (FTD-s) disorders, including FTD, progressive supranuclear palsy (PSP) and corticobasal syndrome, and a common extended haplotype spanning across the MAPT locus is associated with increased risk of PSP and Parkinson's disease. We identified a rare tau variant (p.A152T) in a patient with a clinical diagnosis of PSP and assessed its frequency in multiple independent series of patients with neurodegenerative conditions and controls, in a total of 15 369 subjects. Tau p.A152T significantly increases the risk for both FTD-s (n = 2139, OR = 3.0, CI: 1.6-5.6, P = 0.0005) and Alzheimer's disease (AD) (n = 3345, OR = 2.3, CI: 1.3-4.2, P = 0.004) compared with 9047 controls. Functionally, p.A152T (i) decreases the binding of tau to microtubules and therefore promotes microtubule assembly less efficiently; and (ii) reduces the tendency to form abnormal fibers. However, there is a pronounced increase in the formation of tau oligomers. Importantly, these findings suggest that other regions of the tau protein may be crucial in regulating normal function, as the p.A152 residue is distal to the domains considered responsible for microtubule interactions or aggregation. These data provide both the first genetic evidence and functional studies supporting the role of MAPT p.A152T as a rare risk factor for both FTD-s and AD and the concept that rare variants can increase the risk for relatively common, complex neurodegenerative diseases, but since no clear significance threshold for rare genetic variation has been established, some caution is warranted until the findings are further replicated.

Rare mutations in AβPP, PSEN1, and PSEN2 cause uncommon early onset forms of Alzheimer's disease (AD), and common variants in MAPT are associated with risk of other neurodegenerative disorders. We sought to establish whether common genetic variation in these genes confer risk to the common form of AD which occurs later in life (>65 years). We therefore tested single-nucleotide polymorphisms at these loci for association with late-onset AD (LOAD) in a large case-control sample consisting of 3,940 cases and 13,373 controls. Single-marker analysis did not identify any variants that reached genome-wide significance, a result which is supported by other recent genome-wide association studies. However, we did observe a significant association at the MAPT locus using a gene-wide approach (p = 0.009). We also observed suggestive association between AD and the marker rs9468, which defines the H1 haplotype, an extended haplotype that spans the MAPT gene and has previously been implicated in other neurodegenerative disorders including Parkinson's disease, progressive supranuclear palsy, and corticobasal degeneration. In summary common variants at AβPP, PSEN1, and PSEN2 and MAPT are unlikely to make strong contributions to susceptibility for LOAD. However, the gene-wide effect observed at MAPT indicates a possible contribution to disease risk which requires further study.

Recent large-scale genetic studies of late-onset Alzheimer's disease have identified risk variants in CALHM1, GAB2, and SORL1. The mechanisms by which these genes might modulate risk are not definitively known. CALHM1 and SORL1 may alter amyloid-β (Aβ) levels and GAB2 may influence phosphorylation of the tau protein. In this study we have analyzed disease associated genetic variants in each of these genes for association with cerebrospinal fluid (CSF) Aβ or tau levels in 602 samples from two independent CSF series. We failed to detect association between CSF Aβ42 levels and single nucleotide polymorphisms in SORL1 despite substantial statistical power to detect association. While we also failed to detect association between variants in GAB2 and CSF tau levels, power to detect this association was limited. Finally, our data suggest that the minor allele of rs2986017, in CALHM1, is marginally associated with CSF Aβ42 levels. This association is consistent with previous reports that this non-synonymous coding substitution results in increased Aβ levels in vitro and provides support for an Aβ-related mechanism for modulating risk for Alzheimer's disease.

We undertook a two-stage genome-wide association study (GWAS) of Alzheimer's disease (AD) involving over 16,000 individuals, the most powerful AD GWAS to date. In stage 1 (3,941 cases and 7,848 controls), we replicated the established association with the apolipoprotein E (APOE) locus (most significant SNP, rs2075650, P = 1.8 × 10−157) and observed genome-wide significant association with SNPs at two loci not previously associated with the disease: at the CLU (also known as APOJ) gene (rs11136000, P = 1.4 × 10−9) and 5′ to the PICALM gene (rs3851179, P = 1.9 × 10−8). These associations were replicated in stage 2 (2,023 cases and 2,340 controls), producing compelling evidence for association with Alzheimer's disease in the combined dataset (rs11136000, P = 8.5 × 10−10, odds ratio = 0.86; rs3851179, P = 1.3 × 10−9, odds ratio = 0.86).

This study sets out to identify novel susceptibility genes for late-onset Alzheimer's disease (LOAD) in a powerful set of samples from the UK and USA (1808 LOAD cases and 2062 controls). Allele frequencies of 17 343 gene-based putative functional single nucleotide polymorphisms (SNPs) were tested for association with LOAD in a discovery case-control sample from the UK. A tiered strategy was used to follow-up significant variants from the discovery sample in four independent sample sets. Here, we report the identification of several candidate SNPs that show significant association with LOAD. Three of the identified markers are located on chromosome 19 (meta-analysis: full sample P = 6.94E - 81 to 0.0001), close to the APOE gene and exhibit linkage disequilibrium (LD) with the APOEepsilon4 and epsilon2/3 variants (0.09 < D'

Genetic factors play an important role in the etiology of late-onset Alzheimer's disease (LOAD). We tested gene-centric single nucleotide polymorphisms (SNPs) on chromosome 9 and identified two SNPs in the death-associated protein kinase, DAPK1, that show significant association with LOAD. SNP rs4878104 was significantly associated with LOAD in our discovery case-control sample set (WU) and replicated in each of two initial validation case-control sample sets (P

Yonghong Li, Andrew Grupe, Charles Rowland, Petra Nowotny, John S.K. Kauwe, Scott Smemo, Anthony Hinrichs, Kristina Tacey, Shirley Kwok, Joseph Catanese, John Sninsky, Thomas J. White, Paul Hollingworth, Sandra L. Harris, Arnold Levine, Fabienne Wavrant-De Vrieze, John Hardy, Michael O’Donovan, Simon Lovestone, John Morris, Leon J. Thal, Michael Owen, Julie Williams, Alison Goate, Celera Diagnostics, Alameda, CA, USA; Washington University, St. Louis, MO, USA; Cardiff University, Cardiff, United Kingdom; Robert Wood Johnson Medical School, New Brunswick, NJ, USA; Institute for Advanced Study, Princeton, NJ, USA; National Institute on Aging, Bethesda, MD, USA; Institute of Psychiatry, London, United Kingdom; University of California, San Diego, San Diego, CA, USA. Contact e-mail: yonghong.li@celeradiagnostics.com

Genetic analyses in Caenorhabditis elegans demonstrate that sel-12 and hop-1 , homologues of the Alzheimer’s disease-associated presenilin genes, modify signaling through LIN-12 and GLP-1, homologues of the Notch cell surface receptor. To gain insight into the biochemical basis of this genetic interaction, we tested the possibility that presenilin-1 (PS1) physically associates with the Notch1 receptor in mammalian cells. Notch1 and PS1 coimmunoprecipitated from transiently transfected human embryonic kidney 293 cell lysates in a detergent-sensitive manner, consistent with a noncovalent physical association between the two proteins. The interaction predominantly occurred early in the secretory pathway prior to Notch cleavage in the Golgi, because PS1 immunoprecipitation preferentially recovered the full-length Notch1 precursor. When PS1 was immunoprecipitated from 293 cells that had been metabolically labeled with [ 35 S]methionine and [ 35 S]cysteine, Notch1 was the primary protein detected in PS1 immunoprecipitates, suggesting that this interaction is specific. Furthermore, endogenous Notch and presenilin coimmunoprecipitated from cultured Drosophila cells, indicating that physical interaction can occur at physiological expression levels. These results suggest that the genetic relationship between presenilins and the Notch signaling pathway derives from a direct physical association between these proteins in the secretory pathway.

Thirteen families have been described with an autosomal dominantly inherited dementia named frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17)(1-9), historically termed Pick's disease(10). Most FTDP-17 cases show neuronal and/or glial inclusions that stain positively with antibodies raised against the microtubule-associated protein Tau, although the Tau pathology varies considerably in both its quantity (or severity) and characteristics(1-8,12). Previous studies have mapped the FTDP-17 locus to a 2-centimorgan region on chromosome 17q21.11; the tau gene also lies within this region. We have now sequenced tau in FTDP-17 families and identified three missense mutations (G272V, P301L and R406W) and three mutations in the 5' splice site of exon in. The splice-site mutations all destabilize a potential stem-loop structure which is probably involved in regulating the alternative splicing of exon10 (ref. 13). This causes more frequent usage of the 5' splice site and an increased proportion of tan transcripts that include exon 10. The increase in exon 10(+) messenger RNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17 (refs 12, 14).

Background Apolipoprotein E (APOE) is the most statistically significant genetic risk factor for late-onset Alzheimer disease (LOAD). The linkage disequilibrium pattern around the APOE gene has made it difficult to determine whether all the association signal is derived from APOE or whether there is an independent signal from a nearby gene. Objective To attempt to replicate a recently reported association of APOE 3–TOMM40 haplotypes with risk and age at onset. Design We used standard techniques to genotype several polymorphisms in the APOE–TOMM40 region in a large case-control series, in a series with cerebrospinal fluid biomarker data, and in brain tissue. Setting Alzheimer's Disease Research Center. Participants Research volunteers who were cognitively normal or had Alzheimer disease. Main Outcome Measures Disease status and age at onset. Results We did not replicate the previously reported association of the polyT polymorphism (rs10524523) with risk and age at onset. We found a significant association between rs10524523 and risk of LOAD in APOE 33 homozygotes but in the opposite direction as the previously reported association (the very long allele was underrepresented in cases vs controls in this study (P = .004]). We found no association between rs10524523 and cerebrospinal fluid tau or β-amyloid 42 levels or TOMM40 or APOE gene expression. Conclusions Although we did not replicate the earlier association between the APOE 3–TOMM40 haplotypes and age at onset, we observed that the polyT polymorphism is associated with risk of LOAD in APOE 33 homozygotes in a large case-control series but in the opposite direction as in the previous study.

Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.

L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60–70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10–30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control portein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.