Genome-wide identification of human functional DNA using a neutral indel model

It has become clear that a large proportion of functional DNA in the human genome does not code for protein. Identification of this non-coding functional sequence using comparative approaches is proving difficult and has previously been thought to require deep sequencing of multiple vertebrates. Here we introduce a new model and comparative method that, instead of nucleotide substitutions, uses the evolutionary imprint of insertions and deletions (indels) to infer the past consequences of selection. The model predicts the distribution of indels under neutrality, and shows an excellent fit to human–mouse ancestral repeat data. Across the genome, many unusually long ungapped regions are detected that are unaccounted for by the neutral model, and which we predict to be highly enriched in functional DNA that has been subject to purifying selection with respect to indels. We use the model to determine the proportion under indel-purifying selection to be between 2.56% and 3.25% of human euchromatin. Since annotated protein-coding genes comprise only 1.2% of euchromatin, these results lend further weight to the proposition that more than half the functional complement of the human genome is non-protein-coding. The method is surprisingly powerful at identifying selected sequence using only two or three mammalian genomes. Applying the method to the human, mouse, and dog genomes, we identify 90 Mb of human sequence under indel-purifying selection, at a predicted 10% false-discovery rate and 75% sensitivity. As expected, most of the identified sequence represents unannotated material, while the recovered proportions of known protein-coding and microRNA genes closely match the predicted sensitivity of the method. The method's high sensitivity to functional sequence such as microRNAs suggest that as yet unannotated microRNA genes are enriched among the sequences identified. Futhermore, its independence of substitutions allowed us to identify sequence that has been subject to heterogeneous selection, that is, sequence subject to both positive selection with respect to substitutions and purifying selection with respect to indels. The ability to identify elements under heterogeneous selection enables, for the first time, the genome-wide investigation of positive selection on functional elements other than protein-coding genes.     

Counting all possible ancestral configurations of sample sequences in population genetics

Given a set D of input sequences, a genealogy for D can be constructed backward in time using such evolutionary events as mutation, coalescent, and recombination. An ancestral configuration (AC) can be regarded as the multiset of all sequences present at a particular point in time in a possible genealogy for D. The complexity of computing the likelihood of observing D depends heavily on the total number of distinct ACs of D and, therefore, it is of interest to estimate that number. For D consisting of binary sequences of finite length, we consider the problem of enumerating exactly all distinct ACs. We assume that the root sequence type is known and that the mutation process is governed by the infinite-sites model. When there is no recombination, we construct a general method of obtaining closed-form formulas for the total number of ACs. The enumeration problem becomes much more complicated when recombination is involved. In that case, we devise a method of enumeration based on counting contingency tables and construct a dynamic programming algorithm for the approach. Last, we describe a method of counting the number of ACs that can appear in genealogies with less than or equal to a given number R of recombinations. Of particular interest is the case in which R is close to the minimum number of recombinations for D.     

Adrenoceptors and signal transduction in neurons

The adrenergic system is an essential regulator of neuronal, endocrine, cardiovascular, vegetative, and metabolic functions. The endogenous catecholamines epinephrine and norepinephrine activate G-protein-coupled receptors to transmit their signal across the plasma membrane. These adrenoceptors can be divided into three different groups: the α₁-receptors (α_1A, α_1B, α_1D), α₂-receptors (α_2A, α_2B, α_2C), and β-receptors (β₁, β₂, β₃). This review summarizes recent findings in the field of adrenoceptor signaling in neurons and includes a discussion of receptor-associated proteins, receptor dimerization, subcellular trafficking, and fluorescence optical methods for studying the kinetics of adrenergic signaling. Spatio-temporal imaging may become an important future tool for identifying the physiological significance of these complex signaling mechanisms in vivo. Gene-targeted mouse models carrying deletions in α₂-adrenoceptor have provided detailed insights into specific neuronal functions of the three α₂-receptor subtypes.     

Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice

The CTG repeat involved in myotonic dystrophy is one of the most unstable trinucleotide repeats. However, the molecular mechanisms underlying this particular form of genetic instability—biased towards expansions—have not yet been completely elucidated. We previously showed, with highly unstable CTG repeat arrays in DM1 transgenic mice, that Msh2 is required for the formation of intergenerational and somatic expansions. To identify the partners of Msh2 in the formation of intergenerational CTG repeat expansions, we investigated the involvement of Msh3 and Msh6, partners of Msh2 in mismatch repair. Transgenic mice with CTG expansions were crossed with Msh3- or Msh6-deficient mice and CTG repeats were analysed after maternal and paternal transmissions. We demonstrated that Msh3 but not Msh6 plays also a key role in the formation of expansions over successive generation. Furthermore, the absence of one Msh3 allele was sufficient to decrease the formation of expansions, indicating that Msh3 is rate-limiting in this process. In the absence of Msh6, the frequency of expansions decreased only in maternal transmissions. However, the significantly lower levels of Msh2 and Msh3 proteins in Msh6 -/- ovaries suggest that the absence of Msh6 may have an indirect effect.     

A yeast-based model of alpha-synucleinopathy identifies compounds with therapeutic potential

We have developed a yeast-based model recapitulating neurotoxicity of alpha-synuclein fibrilization. This model recognized metal ions, known risk factors of alpha-synucleinopathy, as stimulators of alpha-synuclein aggregation and cytotoxicity. Elimination of Yca1 caspase activity augmented both cytotoxicity and inclusion body formation, suggesting the involvement of apoptotic pathway components in toxic alpha-synuclein amyloidogenesis. Deletion of hydrophobic amino acids at positions 66-74 in alpha-synuclein reduced its cytotoxicity but, remarkably, did not lower the levels of insoluble alpha-synuclein, indicating that noxious alpha-synuclein species are different from insoluble aggregates. A compound screen aimed at finding molecules with therapeutic potential identified flavonoids with strong activity to restrain alpha-synuclein toxicity. Subsequent structure-activity analysis elucidated that these acted by virtue of anti-oxidant and metal-chelating activities. In conclusion, this yeast-cell model as presented allows not only fundamental studies related to mechanisms of alpha-synuclein-instigated cellular degeneration, but is also a valid high-throughput identification tool for novel neuroprotective agents.     

Chlorodifluoromethyl-substituted monosaccharide derivatives--radical activation of the carbon-chlorine-bond

The dithionite-mediated addition of BrCF(2)Cl to 3,4-di-O-pivaloyl-D-xylal (1) generated preferably 1-CF(2)Cl-substituted products, that is, (2-bromo-2-deoxy-3,4-di-O-pivaloyl-beta-D-xylopyranosyl)-chlorodifluoromethane and (2-deoxy-3,4-di-O-pivaloyl-beta-D-threo-pentopyranosyl)-chlorodifluoromethane. Selected chlorodifluoromethyl-substituted monosaccharide derivatives were hydrodechlorinated or alkylated at the CF(2)Cl-group using tin reagents under radical reaction conditions. Thus, hydrodechlorinations of (2,3,4-tri-O-acetyl-6-deoxy-alpha-L-galactopyranosyl)-chlorodifluoromethane and of methyl 3,4-di-O-acetyl-2-C-chlorodifluoromethyl-2,6-dideoxy-alpha/beta-L-glucopyranoside are reported using tri-n-butyltin hydride initiated by AIBN. UV-initiated allylations are reported for reactions of (2-deoxy-3,4-di-O-pivaloyl-beta-D-threo-pentopyranosyl)-chlorodifluoromethane, (2,3,4-tri-O-acetyl-6-deoxy-alpha-L-galactopyranosyl)-chlorodifluoromethane, 1,3,4,6-tetra-O-acetyl-2-C-chlorodifluoromethyl-2-deoxy-alpha-D-glucopyranose, 1,3,4,6-tetra-O-acetyl-2-C-chlorodifluoromethyl-2-deoxy-alpha-D-mannopyranose and methyl 3,4-di-O-acetyl-2-C-chlorodifluoromethyl-2-deoxy-alpha/beta-D-rabinopyranoside with allyltri-n-butyltin.     

Additive roles of XPA and MSH2 genes in UVB-induced skin tumorigenesis in mice

We have made xeroderma pigmentosum group A gene (XPA)-knockout mice (XPA(-/-) mice). The XPA(-/-) mice had no detectable activity for nucleotide excision repair (NER) and showed a high incidence of UVB-induced skin tumorigenesis. We have also found that cell lines derived from skin cancers in UVB-irradiated XPA(-/-) mice become tolerant to UV-irradiation and showed abnormal UV-induced cell cycle checkpoints and decreased mismatch repair (MMR) activity. These results suggested that the MMR-downregulation may help cells escape killing by UV-irradiation and thus MMR-deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells. In this report, we examined whether the incidence of UVB-induced skin tumorigenesis is enhanced in XPA(-/-)MSH2(-/-), XPA(-/-) and MSH2(-/-) mice when compared with that in wild-type mice. Our results indicate that the MSH2-deficiency caused a high incidence of spontaneous and UVB-induced skin tumorigenesis and the XPA and MSH2 genes have additive roles in the UV-induced skin tumorigenesis.     

Search for clastogenic factors in the plasma of locally irradiated individuals

In studies reported in the 1960s and in several investigations since, plasma from irradiated individuals was shown to induce chromosomal aberrations when transferred into normal blood cultures. In the present study, the aim was to investigate the occurrence of these clastogenic factors (CF) using markers representing DNA damage produced in reporter lymphocytes that are treated with plasma from locally exposed individuals. Blood plasma was obtained from clinical patients with benign conditions before and after they had received radiation to small treatment volumes. Three patient groups were studied: (I) marginal resected basal cell carcinoma, (II) painful osteoarthritis of the knee, and (III) painful tendinitis of the elbow or the heel. Patients in each treatment group obtained the same fractionated treatment regimen, ranging from a total dose of 40 Gy (8 × 5 Gy, 2 factions/week) to a very small volume (1-3.5 cm3) in group I to a total dose of 6 Gy (6 × 1 Gy, 2 fractions/week) for groups II and III (treatment volumes 800-1150 cm3 and 80-160 cm3, respectively). The presence of CF in the plasma was investigated through cytogenetic (chromosomal aberrations, micronuclei) assays and kinetics of early DNA damage (γ-H2AX foci) in reporter cells. With the experimental settings applied, local radiation exposure had no apparent effect on the induction of CF in patient plasma; no deviations in chromosomal aberrations or micronucleus or focus induction were observed in reporter cells treated with postexposure plasma with respect to pre-exposure samples when the mean values of the groups were compared. However, there was a large interindividual variation in the plasma-induced DNA-damaging effects. Steroid treatment of patients was demonstrated to be the most influential factor affecting the occurrence of plasma factors; plasma from patients treated with steroids led to significant reductions of γ-H2AX foci and reduced numbers of chromatid aberrations in reporter cells. In addition to the locally exposed patients, newly obtained plasma samples from three radiological accident victims exposed in 1994 were examined. In contrast to the patient data, a significant increase in chromosomal aberrations was induced with plasma from two accident victims.     

Epigenetic variation during the adult lifespan: cross-sectional and longitudinal data on monozygotic twin pairs

The accumulation of epigenetic changes was proposed to contribute to the age-related increase in the risk of most common diseases. In this study on 230 monozygotic twin pairs (MZ pairs), aged 18-89 years, we investigated the occurrence of epigenetic changes over the adult lifespan. Using mass spectrometry, we investigated variation in global (LINE1) DNA methylation and in DNA methylation at INS, KCNQ1OT1, IGF2, GNASAS, ABCA1, LEP, and CRH, candidate loci for common diseases. Except for KCNQ1OT1, interindividual variation in locus-specific DNA methylation was larger in old individuals than in young individuals, ranging from 1.2-fold larger at ABCA1 (P = 0.010) to 1.6-fold larger at INS (P = 3.7 × 10(-07) ). Similarly, there was more within-MZ-pair discordance in old as compared with young MZ pairs, except for GNASAS, ranging from an 8% increase in discordance each decade at CRH (P = 8.9 × 10(-06) ) to a 16% increase each decade at LEP (P = 2.0 × 10(-08) ). Still, old MZ pairs with strikingly similar DNA methylation were also observed at these loci. After 10-year follow-up in elderly twins, the variation in DNA methylation showed a similar pattern of change as observed cross-sectionally. The age-related increase in methylation variation was generally attributable to unique environmental factors, except for CRH, for which familial factors may play a more important role. In conclusion, sustained epigenetic differences arise from early adulthood to old age and contribute to an increasing discordance of MZ twins during aging.