Conformational properties of DNA strands containing guanine-adenine and thymine-adenine repeats

CD spectroscopy and PAGE were used to cooperatively analyze melting conformers of DNA strands containing GA and TA dinucleotide repeats. The 20mer (GA)10 formed a homoduplex in neutral solutions containing physiological concentrations of salts and this homoduplex was not destabilized even in the terminal (GA)3 hexamers of (GA)3(TA)4(GA)3, although the central (TA)4 portion of this oligonucleotide preserved the conformation adopted by (TA)10. This observation demonstrates that homoduplexes of alternating GA and TA sequences can co-exist in a single DNA molecule. Another 20mer, (GATA)5, adopted as a whole either the AT duplex, like (TA)10, or the GA duplex, like (GA)10, and switched between them reversibly. The concentration of salt controlled the conformational switching. Hence, guanine and thymine share significant properties regarding complementarity to adenine, while the TA and GA sequences can stack in at least two mutually compatible ways within the DNA duplexes analyzed here. These properties extend our knowledge of non-canonical structures of DNA.     

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.     

Evolutionary dynamics of tandem repeats in the mitochondrial DNA control region of the minnow Cyprinella spiloptera

Length variation due to tandem repeats is now recognized as a common feature of animal mitochondrial DNA; however, the evolutionary dynamics of repeated sequences are not well understood. Using phylogenetic analysis, predictions of three models of repeat evolution were tested for arrays of 260-bp repeats in the cyprinid fish Cyprinella spiloptera. Variation at different nucleotide positions in individual repeats supported different models of repeat evolution. One set of characters included several nucleotide variants found in all copies from a limited number of individuals, while the other set included an 8-bp deletion found in a limited number of copies in all individuals. The deletion and an associated nucleotide change appear to be the result of a deterministic, rather than stochastic, mutation process. Parallel origins of repeat arrays in different mitochondrial lineages, possibly coupled with a homogenization mechanism, best explain the distribution of nucleotide variation.     

Comparison of the solution structures of intramolecular DNA triple helices containing adjacent and non-adjacent CG.C+ triplets

The solution conformations of the intramolecular triple helices d(AGAAGA-X-TCTTCT-X-TC+TTC+T) and d(AAGGAA-X-TTCCTT-X-TTC+C+TT) (X = non-nucleotide linker) have been determined by NMR.1H NMR spectra in H2O showed that the third strand cytosine residues are fully paired with the guanine residues, each using two Hoogsteen hydrogen bonds. Determination of the13C chemical shifts of the cytosine C6 and C5 and their one-bond coupling constants (1 J CH) conclusively showed that the Hoogsteen cytosine residues are protonated at N3. The global conformations of the two molecules determined with >19 restraints per residue are very similar (RMSD = 0.96 A). However, some differences in local conformation and dynamics were observed for the central two base triplets of the two molecules. The C N3H were less labile in adjacent CG.C+triplets than in non-adjacent ones, indicating that the adjacent charge does not kinetically destabilize these triplets. The sugar conformations of the two adjacent cytosine residues were different and the 5'-residue was atypical of protonated cytosine. Hence, there are subtle effects of the interaction between two adjacent cytosine residues. The central two purines in each sequence showed non-standard backbone conformations, averaging between gamma approximately 60 degrees and gamma approximately 180 degrees. This may be related to the difference in the dependence of the thermodynamic stability on pH observed for these two sequences.     

Mitochondrial DNA variable number tandem repeats (VNTRs): utility and problems in molecular ecology

Analysis of mitochondrial (mt)DNA size polymorphism in the form of variable number tandem repeats (mtVNTRs) has become an increasingly popular methodology for addressing questions in molecular ecology. When detected by PCR, mtVNTR analysis can provide a sensitive, rapid, and cost-effective measure of genetic variability that may be exploited in studies of population differentiation and biogeography. Despite the emergence of this approach, there has been little critical evaluation of its success or utility as a practical tool. In this review, we identify problematic methodological, theoretical and interpretive factors that can influence the utility of mtVNTR analysis. The reliability of the procedure is considered in terms of both detection of alleles and scoring of intra-individual allele frequencies. While many of the potential technical problems of the technique do not raise serious practical concerns, this rapid and sensitive methodology is seriously compromised by the difficulty of reliably assessing allele frequencies, of assaying only germline tissue, and in our ignorance of the mechanisms generating mtVNTR diversity. Thus, although there is a considerable potential for mtVNTR pilot studies to assess genetic diversity, the utility of the technique to resolve broader questions in molecular ecology should be treated cautiously until such a time as the system is better understood.     

Evaluation of oligonucleotide probes for simple tandem repeats (STR) to produce informative DNA fingerprints of the chicken

Early intervention programs are designed to enhance the developmental competence of participants and to prevent or minimize developmental delays. Children targeted for early intervention may either include environmentally or biologically vulnerable children, or those with established developmental deficits. There is growing consensus based on the best available evidence that early interventions can exert moderate positive effects. However, this literature is limited by substantial methodological weaknesses in most studies. Therefore further randomized clinical trials are needed to ascertain which programs best meet the needs of children with or at risk for developmental disability.     

A K cation-induced conformational switch within a loop spanning segment of a DNA quadruplex containing G-G-G-C repeats

We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.     

Differential display of genome subsets containing specific interspersed repeats

A genomic differential display method was developed that analyzes many restriction fragment length polymorphisms simultaneously. Interspersed repeat sequences were used to reduce DNA sample complexity and to target genomic subsets of interest. This work focused on trinucleotide repeats because of their importance in human inherited diseases. Immobilized repeat-containing oligonucleotides were used to capture genomic DNA fragments containing sequences complementary to the oligonucleotide. Captured fragments were amplified by PCR and fluorescently labeled using primers complementary to the repeat sequence and/or to the known sequences ligated to the ends of the restriction fragments. The labeled PCR fragments were displayed by size on a high-resolution automated fluorescent DNA sequencing instrument. Although there was a conservation in the overall pattern of displayed genome subsets, many clear and reproducible differences were detected when genomes from different individuals were compared. Fewer differences were detected within, than between, monozygotic twin pair genomes. In control experiments, the method distinguished between Huntington disease alleles with normal and expanded CAG repeat lengths.     

Acidic peptide-mediated expression of the antimicrobial peptide buforin II as tandem repeats in Escherichia coli

OBJECTIVES: To identify the causes of and discuss nursing management of those complications that are unique to the brain tumor patient population. DATA SOURCES: Published articles and books related to neurological and nonneurological complications in the neuro-oncology patient. CONCLUSIONS: Seizures, cerebral edema, thromboembolism, and endocrine dysfunction are several complications experienced by the neuro-oncology patient. These complications can be caused by the tumor or treatment of this disease. IMPLICATIONS FOR NURSING PRACTICE: Care of the neuro-oncology patient is complex. Knowledge of the potential complications that patients may experience and how to manage these problems serves to enhance their quality of life.     

Conformational analyses of somatostatin-related cyclic hexapeptides containing peptoid residues

We report the conformational analysis by 1H NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of a series of peptoid analogues of the cyclic hexapeptide c[Phe11-Pro6-Phe7-d-Trp8-Lys9-Thr10] (1). The proline residue in compound 1 is replaced with the peptoid residues N-benzylglycine (Nphe) (compound 2), N-(S)-alpha-methylbenzylglycine [(S)-beta-MeNphe] (compound 3), and N-(R)-alpha-methylbenzylglycine [(R)-beta-MeNphe] (compound 4). The peptoid analogues 2 and 4 exhibit potent binding activities to the hsst2 receptor, while the binding affinities to the hsst5 and to the hsst3 receptors are reduced compared to that of the parent compound 1. Compound 3 shows reduced binding activities to the hsst2, hsst3, and hsst5 receptors compared to compound 1. The results of in vivo assays indicate that these compounds inhibit the growth hormone release but do not affect the insulin release. These peptoid-containing analogues show two sets of NMR signals corresponding to cis and trans conformations of the peptide bond between Phe11 and Nxaa6. We demonstrate that the backbone conformation and the orientation of the relevant side chains of compound 1 are maintained in the cis isomers of the peptoid analogues which adopt a type VI beta-turn centered around residues 11 and 6 and a type II' beta-turn with d-Trp in the i+1 position. The enhanced selectivity of the peptoid-containing analogues compared to compound 1 and the results of the conformational analysis suggest that the presence of a conformationally constrained hydrophobic group in position 6 in complementary topology to the Phe11 side chain enhances selective binding to the hsst2 receptor.     

Molecular characterization of a Plasmodium chabaudi erythrocyte membrane-associated protein with glutamate-rich tandem repeats

BACKGROUND: The fourth American College of Chest Physicians Consensus Conference on Antithrombotic Therapy recently published guidelines that included recommendations regarding the management of excessive anticoagulation. Limited data are available to support these recommendations. OBJECTIVES: To assess management and outcomes of excessive anticoagulation in a group model health maintenance organization, compare management with the published guidelines, and analyze the cost of treatment strategies. METHODS: A search of computerized laboratory information identified patients with an international normalized ratio (INR) of greater than 6.0 during the 9-month study. Pertinent data were collected through a retrospective medical record review. Information was concurrently collected for cost analyses. RESULTS: The analysis included 301 episodes of excessive anticoagulation among 248 patients. Most (83%) episodes of elevated INRs were managed conservatively by a temporary discontinuation of warfarin sodium therapy until the INR was in a therapeutic range. Conservative management resulted in no sequelae in 212 (85.1%) of 249 episodes. Two episodes (0.8%) of major bleeding evolved in patients managed conservatively. No sequelae were documented in 23 (44%) of 52 episodes of phytonadione (vitamin K1) administration. Sixteen (31%) episodes of major bleeding were documented, but bleeding occurred before phytonadione administration in all cases. Administering phytonadione resulted in hospital admission for 3 patients--2 (3.8%) because of thromboembolism and 1 (1.9%) for the administration of heparin sodium. Cost-effectiveness analysis determined that treatment with phytonadione is 7 times more costly than conservative management when INRs are between 6.0 and 10.0. CONCLUSIONS: Most episodes of excessive anticoagulation were not managed per consensus guidelines. The higher the INR, the more likely were interventions to adhere to the guidelines. Administering phytonadione to patients with a moderate elevation of INRs (6.0-10.0) may be unnecessary. Based on this study, conservative management is a viable option.     

The emergence of new DNA repeats and the divergence of primates

We have identified four genetic novelties that are fixed in specific primate lineages and hence can serve as phylogenetic time markers. One Alu DNA repeat is present in the human lineage but is absent from the great apes. Another Alu DNA repeat is present in the gorilla lineage but is absent from the human, chimpanzee, and orangutan. A progenitor Xba1 element is present in the human, chimpanzee, gorilla, and orangutan, but only in the human lineage did it give rise to a transposed progeny, Xba2. The saltatory appearance of Xba2 is an example of a one-time event in the evolutionary history of a species. The enolase pseudogene, known to be present as a single copy in the human, was found to be present in four other primates, including the baboon, an Old World monkey. Using the accepted value of 5 x 10(-9) nucleotide substitutions per site per year as the evolutionary rate for pseudogenes, we calculated that the enolase pseudogene arose approximately 14 million years ago. The calculated age for this pseudogene and its presence in the baboon are incongruent with each other, since Old World monkeys are considered to have diverged from the hominid lineage some 30 million years ago. Thus the rate of evolution in the enolase pseudogene is only about 2.5 x 10(-9) substitutions per site per year, or half the rate in other pseudogenes. It is concluded that rates of substitution vary between species, even for similar DNA elements such as pseudogenes. We submit that new DNA repeats arise in the genomes of species in irreversible and punctuated events and hence can be used as molecular time markers to decipher phylogenies.     

A dedicated internal standard in fragment length analysis of hyperpolymorphic short tandem repeats

Some of the most polymorphic short tandem repeats (STRs) have alleles differing in size by as little as one basepair. Since sizing precision with commercially available internal standards ordinarily does not allow safe discrimination at this level, typing is accomplished through comparisons with allelic ladders run on each gel. Here we introduce the use of optimally spaced sequenced alleles as a dedicated internal standard for measurement and typing of hyperpolymorphic STRs. We have constructed such a dedicated internal standard for HUMACTBP2 ([1]; Moos and Gallwitz, EMBO I., 5 (1983) 757-761) typing, including 25 sequenced, ROX labelled HUMACTBP2 alleles spanning the size range for alleles at this locus (233-333 basepairs (bp)). Comparisons of inter-gel runs of selected alleles with this dedicated standard and the commercial GS500 internal standard demonstrate that only the presently described internal standard is suited for direct typing based on fragment length measurements only. Obvious advantages of this typing procedure are that fragment measurements are precise and in accordance with sequenced lengths, and that the typing procedure via an external allelic ladder is avoided. Sequences of the alleles used in the internal standard as well as of selected alleles for allelic ladders in two other hyperpolymorphic AAAG-repeat STRs, D11S554 (Phromchotikul et al., Hum. Mol. Genet., 3 (1991) 21) and HUMAPOA11 (Kimpton et al., PCR Methods Appl., 3 (1993) 13-22) are also presented. To allow fragment length analysis of these two STRs, five D11S554 alleles, spanning from 176 to 225 basepairs, were added to the dedicated internal standard. Performing similar comparisons as for HUMACTBP2, it is demonstrated that even if sizing accuracy is not as good as with HUMACTBP2, typing based on measured fragment size only may be achieved with both other STRs as well. Selecting different colours for the three STRs, triplex electrophoretic runs were performed of a Norwegian population database of 300 unrelated individuals. The probability that two unrelated individuals have the same type in all three STRs is 5 x 10(-7), and the combined paternity exclusion power 99.77%, indicating that this STR selection may represent a good choice for forensic genetic casework.     

Stabilization of the purine.purine.pyrimidine DNA base triplets by divalent metal cations

An extensive search of aphasia-treatment literature yielded 55 reports of clinical outcomes satisfying the essential criteria for inclusion in a meta-analysis. The results confirmed those of an earlier meta-analysis in demonstrating the utility of aphasia treatments, generally considered, for bringing about desirable clinical outcomes. Beyond the general case, the new findings address clinical utility in finer detail than was previously possible. Effects of treatment for aphasia are synthesized and assessed for each of four important dimensions: amount of treatment, type of treatment, severity of aphasia, and type of aphasia.     

The variable number of tandem repeats in the renin gene of rats and possible significance in hypertension

The variable number of tandem repeats (VNTR) in the first intron of renin gene for the spontaneously hypertensive rat (SHR), its controls Wistar-Kyoto (WKY), renal hypertensive rat, and Sprague-Dawley rat (SD) were compared by polymerase chain reaction (PCR) method. An analysis of VNTR from WKY, Wistar and SD showed that there are two different renin gene alleles and three genetypes 2.0kb/2.0kb, 2.0kb/1.8kb, 1.8kb/1.8kb. The genetype from renal hypertensive rats is same as those seen in the normal controls. However, compared with the WKY, Wistar and SD genes, a "deletion" of approximately 1.0kb was found in the first intron of the SHR renin gene. Our results strongly suggest that the cause and mechanism of elevated blood pressure is complex, and the molecular basis of the genetic-prone hypertension is existed.     

Relative stability of triplexes containing different numbers of T.AT and C+.GC triplets

We have used DNase I footprinting to compare the stability of parallel triple helices containing different numbers of T.AT and C+. GC triplets. We have targeted a fragment containing the 17mer sequence 5'-AGGAAGAGAAAAAAGAA with the 9mer oligonucleotides 5'-TCCTTCTCT, 5'-TTCTCTTTT and 5'-TTTTTTCTT, which form triplexes at the 5'-end, centre and 3'-end of the target site respectively. Quantitative DNase I footprinting has shown that at pH 5.0 the dissociation constants of these oligonucleotides are 0.13, 4.7 and >30 microM respectively, revealing that increasing the proportion of C+.GC triplets increases triplex stability. The results suggest that the positive charge on the protonated cytosine contributes to triplex stability, either by a favourable interaction with the stacked pisystem or by screening the charge on the phosphate groups. In the presence of a naphthylquinoline triplex binding ligand all three oligonucleotides bind with similar affinities. At pH 6.0 these triplexes only form in the presence of the triplex binding ligand, while at pH 7.5 footprints are only seen with the oligonucleotide which generates the fewest number of C+.GC triplets (TTTTTTCTT) in the presence of the ligand.     

Molecular cloning and expression of a novel human cDNA containing CAG repeats

A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.     

Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage

Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage. One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA. In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates. The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported.