The complex pathology of trinucleotide repeats

The expansion of trinucleotide repeat sequences has now been shown to be the underlying cause of at least ten human disorders. Unifying features among these diseases include the unstable behavior of the triplet repeat during germline transmission when the length of the repeat exceeds a critical value. However, the trinucleotide repeat disorders can be divided into two distinct groups. Type I disorders involve the expansion of CAG repeats, which encode an expanded polyglutamine, inserted into the open-reading frame of a gene that is usually quite broadly expressed. Recently, mouse models for type I disorders have been developed and the basis of pathology is under study, both in these models and through biochemical and cell biological approaches. The type II disorders involve repeat expansions in noncoding regions of genes. The mechanisms by which these repeat expansions lead to pathology may be quite diverse.     

Structure-affinity relationships for the binding of actinomycin D to DNA

Molecular models of the complexes between actinomycin D and 14 different DNA hexamers were built based on the X-ray crystal structure of the actinomycin-d(GAAGCTTC)2 complex. The DNA sequences included the canonical GpC binding step flanked by different base pairs, nonclassical binding sites such as GpG and GpT, and sites containing 2,6-diamino-purine. A good correlation was found between the intermolecular interaction energies calculated for the refined complexes and the relative preferences of actinomycin binding to standard and modified DNA. A detailed energy decomposition into van der Waals and electrostatic components for the interactions between the DNA base pairs and either the chromophore or the peptidic part of the antibiotic was performed for each complex. The resulting energy matrix was then subjected to principal component analysis, which showed that actinomycin D discriminates among different DNA sequences by an interplay of hydrogen bonding and stacking interactions. The structure-affinity relationships for this important antitumor drug are thus rationalized and may be used to advantage in design of novel sequence-specific DNA-binding agents.     

Unstable trinucleotide repeats and the diagnosis of neurodegenerative disease

Human genes containing unstable triplet repeats are associated with several neuropsychiatric diseases. These diseases all show a marked variation in clinical symptoms and genetic anticipation. The molecular understanding of these diseases provides the diagnostic laboratory with the capability to test directly whether an individual's DNA contains the disease-causing mutation, either as a confirmation of a clinical diagnosis or presymptomatically.     

Redistribution of ribosomal DNA after blocking of transcription induced by actinomycin D

In recent years there has been increased interest in the role of exercise in the alleviation of patellofemoral pain syndrome. Contradictions which result in a lack of a consensus amongst clinicians and researchers as to the most beneficial type of exercise are reviewed. The popularly held belief that there are imbalances between some components of the quadriceps femoris is also reviewed and conflicting evidence that quadriceps contractions can be enhanced by altering hip and knee positions is found. Some traditional exercises, previously considered essential to any regime for this common condition, were not found to be as beneficial as previously thought. Consequently, some of these exercises should be less prominent in a rehabilitation programme for patellofemoral pain syndrome.     

Exclusion of expansion of 50 CAG/CTG trinucleotide repeats in bipolar disorder

OBJECTIVE: The purpose of this study was to identify the specific expanded CAG/CTG trinucleotide repeat associated with bipolar disorder. METHOD: The study employed an efficient multistage approach for using a genomic CAG/CTG screening set. RESULTS: The authors found no evidence of expanded repeats at 43 polymorphic autosomal loci and seven X chromosomal loci. Secondary screening was pursued at the only locus that contained a large allele (37 repeats) in the primary screening. No association was found between allele size and diagnostic status. CONCLUSIONS: It is highly unlikely that expansions in repeat size at any of the 50 candidate trinucleotide repeat loci examined are responsible for the association between expanded CAG/ CTG repeats and bipolar disorder. However, although the authors prioritized the repeats that were a priori most likely to be involved, the study does not reject the more general hypothesis that expanded CAG/CTG repeats are implicated in the pathogenesis of bipolar disorder.     

Characterization of Alu repeats that are associated with trinucleotide and tetranucleotide repeat microsatellites

The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats. In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers. Sequences containing Alu repeats were eliminated because unique primers could not be designed. Various classes of microsatellites are associated with different classes of Alu repeats. Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alu repeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY. Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites. Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes. We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study. We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones. Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats. Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from approximately 50% to 10%. The new Alu probes that were designed have also proved to be useful in Alu-Alu fingerprinting.     

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure-specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.     

Three CAG trinucleotide repeats on chromosome 6 (D6S1014, D6S1015, and D6S1058) are not expanded in 30 families with schizophrenia

Despite its importance and widespread usage, the term mixed dementia, referring to the coexistence of Alzheimer's disease (AD) and vascular dementia (VsD), has been ill-defined and poorly conceptualized. The authors review the use of the term mixed dementia in neuropathological and clinical research. As a result of recent developments in the categorization of dementias, they recommend discarding the term mixed dementia in favor of a more precise terminology based on AD and VsD concurrently meeting established criteria for each diagnosis.     

Tandem repeats finder: a program to analyze DNA sequences

A tandem repeat in DNA is two or more contiguous, approximate copies of a pattern of nucleotides. Tandem repeats have been shown to cause human disease, may play a variety of regulatory and evolutionary roles and are important laboratory and analytic tools. Extensive knowledge about pattern size, copy number, mutational history, etc. for tandem repeats has been limited by the inability to easily detect them in genomic sequence data. In this paper, we present a new algorithm for finding tandem repeats which works without the need to specify either the pattern or pattern size. We model tandem repeats by percent identity and frequency of indels between adjacent pattern copies and use statistically based recognition criteria. We demonstrate the algorithm's speed and its ability to detect tandem repeats that have undergone extensive mutational change by analyzing four sequences: the human frataxin gene, the human beta T cellreceptor locus sequence and two yeast chromosomes. These sequences range in size from 3 kb up to 700 kb. A World Wide Web server interface atc3.biomath.mssm.edu/trf.html has been established for automated use of the program.     

Interaction of actinomycin D with suspended mycelium of streptomyces

Binding of exogenous actinomycin D (AMD) by washed mycelium of streptomycetes i.e. variants of Streptomyces chrysomallus producing and not producing actinomycins and Streptomyces lividans not synthesizing the antibiotics was studied. Dependence of the bound quantity of AMD on its concentration, incubation time and temperature, energy source availability, influence of respiration inhibitors and the membranotropic antibiotic gramicidin S was shown. The intracellularly localized portion of the bound AMD likely penetrated to the cells by diffusion and was strongly bound presumably to DNA in the AMD sensitive S.lividans and to the specific intracellular actinomycin-binding proteins in the AMD resistant variants of S.chrysomallus. The ratio of AMD strongly bound by the mycelium and AMD easily washed with physiological solution and probably localized on the surface was determined. The ratio depended on sensitivity of the culture to AMD and for the variants of S.chrysomallus on the age of the culture and its ability to synthesize actinomycins.     

Delocalization of some small nucleolar RNPs after actinomycin D treatment to deplete early pre-rRNAs

Retention of some components within the nucleolus correlates with the presence of rRNA precursors found early in the rRNA processing pathway. Specifically, after most 40S, 38S and 36S pre-rRNAs have been depleted by incubation of Xenopus kidney cells in 0.05 microg/ml actinomycin D for 4 h, only 69% U3 small nucleolar RNA (snoRNA), 68% U14 snoRNA and 72% fibrillarin are retained in the nucleolus as compared with control cells. These nucleolar components are important for processing steps in the pathway that gives rise to 18S rRNA. In contrast, U8 snoRNA, which is used for 5.8S and 28S rRNA production, is fully retained in the nucleolus after actinomycin D treatment. Therefore, U8 snoRNA is in a different category than U3 and U14 snoRNA and fibrillarin. It is proposed that U3 and U14 snoRNA and fibrillarin, but not U8 snoRNA, bind to the external transcribed spacer or internal transcribed spacer 1, and when these binding sites are lost after actinomycin D treatment some of these components cannot be retained in the nucleolus. Other binding sites may also exist, which would explain why only some and not all of these components are lost from the nucleolus.     

Stimulation of leptin release by actinomycin D in rat adipocytes

A greater understanding of the factors causing the enhanced release of leptin by adipocytes in obesity is needed. Experiments were designed to determine the effects of actinomycin D on leptin release by isolated rat adipocytes during primary culture for 24 hr. In adipocytes from fed hypothyroid rats, the initial rate of leptin release over the first 6 hr was not maintained over the next 18 hr. The decline in leptin release by adipocytes in primary culture between 6 and 24 hr was reduced markedly by either dexamethasone or actinomycin D. Both actinomycin D and dexamethasone also reduced the loss of leptin mRNA seen over the 24-hr incubation. Maximal effects on leptin release and leptin mRNA accumulation required only 0.1 microM of actinomycin D, a concentration that had no significant effect on the 18S RNA content of adipocytes at the end of a 24-hr incubation. In contrast to the reduced loss of leptin mRNA seen at 24 hr, the loss of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid (GAPDH mRNA) was enhanced in the presence of 0.1 microM of actinomycin D. The effects of dexamethasone could be differentiated from those of actinomycin D by the finding that cycloheximide blocked the reduced loss of leptin mRNA due to dexamethasone while having no effect on that due to actinomycin D. These results point to a unique regulation of leptin release and leptin mRNA levels by actinomycin D.     

Human stanniocalcin (STC): genomic structure, chromosomal localization, and the presence of CAG trinucleotide repeats

Most anticardiolipin antibodies (ACA) associated with antiphospholipid syndrome (APS) are directed against epitopes expressed on beta2-glycoprotein I (beta2GPI). Despite a good correlation between standard ACA assays and those using purified human beta2GPI as the sole antigen, some sera from APS patients only react in the latter. This is indicative of heterogeneity in anti-beta2GPI antibodies. To characterize their reactivity profiles, human and bovine beta2GPI were immobilized on gamma-irradiated plates (beta2GPI-ELISA), plain polystyrene precoated with increasing cardiolipin concentrations (CL/beta2GPI-ELISA), and affinity columns. Fluid-phase inhibition experiments were also carried out with both proteins. Of 56 selected sera, restricted recognition of bovine or human beta2GPI occurred respectively in 10/29 IgA-positive and 9/22 IgM-positive samples, and most of the latter (8/9) were missed by the standard ACA assay, as expected from a previous study. Based on species specificity and ACA results, IgG-positive samples (53/56) were categorized into three groups: antibodies reactive to bovine beta2GPI only (group I) or to bovine and human beta2GPI, group II being ACA-negative, and group III being ACA-positive. The most important group, group III (n = 33) was characterized by (i) binding when beta2GPI was immobilized on gamma-irradiated polystyrene or cardiolipin at sufficient concentration (regardless of beta2GPI density, as assessed using 125I-beta2GPI); (ii) and low avidity binding to fluid-phase beta2GPI (Kd in the range 10(-5) M). In contrast, all six group II samples showed (i) ability to bind human and bovine beta2GPI immobilized on non-irradiated plates; (ii) concentration-dependent blockade of binding by cardiolipin, suggesting epitope location in the vicinity of the phospholipid binding site on native beta2GPI; (iii) and relative avidities approximately 100-fold higher than in group III. Group I patients were heterogeneous with respect to CL/beta2GPI-ELISA and ACA results (6/14 scored negative), possibly reflecting antibody differences in terms of avidity and epitope specificity. Affinity fractionation of 23 sera showed the existence, in individual patients, of various combinations of antibody subsets solely reactive to human or bovine beta2GPI, together with cross-species reactive subsets present in all samples with dual reactivity namely groups III and II, although the latter antibodies were poorly purified on either column. Therefore, the mode of presentation of beta2GPI greatly influences its recognition by anti-beta2GPI antibodies with marked inter-individual heterogeneity, in relation to ACA quantitation and, possibly, disease presentation and pathogenesis.     

Binding characteristics of SLE anti-DNA autoantibodies to modified DNA analogues

Native calf thymus DNA was modified with furocoumarins and reactive oxygen species (ROS). The modifications were probed by UV & Fluorescence spectroscopy, thermal denaturation and hydroxyapatite chromatography. In CD spectroscopy changes in the ellipticity of the DNA molecule were observed as a result of modification. The binding specificity of naturally occurring anti-DNA antibodies with modified DNA molecules was assessed by ELISA and quantitative precipitin titration. The affinity of anti-DNA antibodies for modified conformers was found to be quite high.     

Predicting sequence-dependent melting stability of short duplex DNA oligomers

Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or tm of short duplex DNA oligomers (approximately 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting tm from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental tm's of short duplex DNA oligomers is assessed.     

Cloned human FMR1 trinucleotide repeats exhibit a length- and orientation-dependent instability suggestive of in vivo lagging strand secondary structure

The normal human FMR1 gene contains a genetically stable (CGG) n trinucleotide repeat which usually carries interspersed AGG triplets. An increase in repeat number and the loss of interspersions results in array instability, predominantly expansion, leading to FMR1 gene silencing. Instability is directly related to the length of the uninterrupted (CGG) n repeat and is widely assumed to be related to an increased propensity to form G-rich secondary structures which lead to expansion through replication slippage. In order to investigate this we have cloned human FMR1 arrays with internal structures representing the normal, intermediate and unstable states. In one replicative orientation, arrays show a length-dependent instability, deletions occurring in a polar manner. With longer arrays these extend into the FMR1 5'-flanking DNA, terminating at either of two short CGG triplet arrays. The orientation-dependent instability suggests that secondary structure forms in the G-rich lagging strand template, resolution of which results in intra-array deletion. These data provide direct in vivo evidence for a G-rich lagging strand secondary structure which is believed to be involved in the process of triplet expansion in humans.     

Simple tandem DNA repeats and human genetic disease

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.     

Method for production of neuronal hybridoma using emetine and actinomycin D

Osteoporosis is a common and costly condition that can cause disability or death. Bone density testing permits identification of individuals who are at high risk of fracture. Adequate calcium, vitamin D, and weight-bearing exercise are important, but pharmacologic intervention is indicated and effective for increasing bone mass and decreasing fracture risk.