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The Alu element, the most prevalent SINE (short interspersed element) in the human genome, is one of the many RNA-encoding genes that evolved from the 7SL RNA gene. During analysis of the evolution of 7SL-derived RNAs, two distinct evolutionary intermediates capable of self-catalyzed DNA depurination (SDP) were identified. These SDP sequences spontaneously create apurinic sites that can result in increased mutagenesis due to their error-prone repair. This DNA self-depurination mechanism has been shown both in vitro and in vivo to lead to substitution and short frameshift mutations at a frequency that far exceeds their occurrence due to random errors in DNA replication. In both evolutionary intermediates, the same self-depurination sequence overlaps motifs necessary for successful transcription and SRP9/14 (signal recognition particle) binding; hence, mutations in this region could disrupt RNA activity. Yet, the 7SL-derived RNAs that arose from the elements capable of SDP show significant diversity in this region, and every new sequence retains the transcription and SRP9/14-binding motifs, even as it has lost the SDP sequence. While some (but not all) of the mutagenesis can be alternatively attributed to CpG decay, the very fact that the self-depurinating sequences are selectively discarded in all cases suggests that this was evolutionarily motivated to prevent further destructive mutagenesis by the SDP mechanism.

Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5′G residue of the 5′GA/TGG loop and of the first 5′A residue of the 5′GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000–4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.

The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze depurination of the 5'G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and β-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to β-globin variants underlying phenotypic diversity and disease.

The sequence variation tolerated within the stem-loop-forming genomic consensus sequence for self-catalyzed site-specific depurination of G residues is explored. The variation in self-depurination kinetics with sequence changes in the loop residues and stem base pairs, as well as with pH, provides insights into the self-catalytic mechanism. The observations suggest that self-catalyzed depurination of the 5' G residue of the loop consensus sequence 5'-G(T/A)GG-3' probably involves formation of some intraloop hydrogen-bonded base pair with the 3'-terminal G residue; although the electronic structure of both these G residues is retained, their 2-amino substituents are not critical for that interaction. The strong dependence of the self-depurination kinetics on stem stability suggests that the lifetime of some strained form of the loop is controlled by the integrity of the stem. In addition to the effects of length and base pair sequence on stem stability, there is a base pair requirement at the base of the loop: self-depurination is suppressed by 5'-C·G-3', 5'-A·T-3', or a mismatch but is most favored by 5'T·A3' and less so by 5'-G·C-3'. The occurrence in T and G of a similarly located carbonyl capable of hydrogen-bonding to the water molecule required for glycosyl bond hydrolysis may explain this sequence requirement. In toto, the more complete definition of the consensus sequence provided by this investigation enables a more accurate estimation of their number in the human genome and their distribution among different genes.

The aim of the present study was to analyze BRCA1 and BRCA2 mutations in Uruguayan families with breast and breast/ovarian cancer. Probands from 42 families with at least three cases of female breast cancer (BC) or two cases and subcriteria (paternal transmission, ovarian cancer, bilateral BC, male BC, Ashkenazi Jewish ancestry) in the same lineage, at least one diagnosed before age 50, were screened for germline mutations. PCR amplification of all exons and intron–exon boundaries were performed, followed by protein truncation test, heteroduplex analysis, and direct sequencing. We identified seven different truncating mutations in seven families, five in BRCA2 (three in site-specific BC families and two in breast–ovarian cancer families) and two in BRCA1 (one in a site-specific BC family and the other in a breast–ovarian cancer family). Both BRCA1 mutations (5583insT and 2687T>G) and one of the five BRCA2 mutations (3829insTdel35) were not previously reported. We also detected ten sequence variants of unknown significance, five of them not described before. The low frequency of BRCA1/2 mutations (0.17) is in agreement with that reported in studies which included families with similar selection criteria. However, the observed predominance of BRCA2 (0.12) over BRCA1 mutations (0.05) is in contrast with the higher proportion of BRCA1 mutations communicated for most previous studies, even those with a predominance of site-specific BC families. Meanwhile, it has been described in one Chilean and some Spanish and Italian reports, highlighting the strong dependence between the mutational spectra and the ethnicity of the population analyzed.

The human β-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5′GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze depurination of the loop 5′G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of β-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and β-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-depurination sites, which differ by only one or two base pairs in the stem region from that of the β-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-depurination site in β-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA depurination associated with variability of mutation incidence among human β-globin paralogs.

Few studies have investigated the frequency of hereditary non-polyposis colorectal cancer (HNPCC) in patients with colorectal cancer (CRC), and these have shown marked geographic variations. The aim of this study was to estimate the frequency of HNPCC in a cohort of Uruguayan CRC patients. We included all patients operated consecutively for CRC in the Hospital Central de las Fuerzas Armadas (Uruguay) between 1987 and 2003. Cases were classified into three groups: (i) those fulfilling Amsterdam criteria; (ii) those not fulfilling Amsterdam criteria but considered as a population at increased risk of cancer; and (iii) sporadic CRC. Genetic analysis to detect point mutations in hMLH/hMSH2/hMSH6 genes was performed in group 1 patients. Cases not showing mutations were tested by multiplex ligation-dependent probe amplification. Among 461 patients, group 1 represented 2.6%, group 2 represented 5.6%, and sporadic cases 91.8%. hMLH1/hMSH2/hMSH6 mutations were found in 25% of cases classified as HNPCC (two in hMLH1 and one in hMSH2). No mutations were detected in hMSH6 gene. The proportion of CRC patients that fulfilled Amsterdam criteria agrees with other reports. However, the percentage of HNPCC cases with identified mutations (25%) may be lower than that reported from other populations. This may reflect, among other possible causes, a different genetic profile in the Uruguayan population.

Mixed epithelial and stromal tumor of the kidney is a rare biphasic tumor composed of cysts and tubules embedded in the spindle cell stroma. Although the histogenesis of this tumor is unknown, it has been proposed that both components of the tumor, i.e., stromal and epithelial, are neoplastic. The authors report preliminary immunohistochemical and electron microscopic studies of the epithelial component from one case of a typical, benign, mixed epithelial, and stromal tumor of the kidney. In this study, some tubules showed positivity for proximal, while others showed positivity for distal, nephron immunomarkers. By electron microscopy, some tubules had features of proximal tubular epithelium, while other tubules had features of the loop of Henle (thin segments). The authors believe that in a benign tumor such morphologic heterogeneity is inconsistent with neoplastic proliferation. Therefore, they postulate that in mixed epithelial and stromal tumor of the kidney the tubules are entrapped rather than neoplastic. Additional studies are needed to address this issue and electron microscopy should play a significant role in this process.

Resumen Introduccion: Los tumores de los cordones sexuales y del estroma corresponden al 5% de los tumores ovaricos. La variedad a celulas de Leydig es relativamente rara y se han descrito pocos casos en la bibliografia. Sin embargo, es uno de los diagnosticos que se debe considerar en todas las pacientes posmenopausicas con signos de virilizacion. Caso clinico: Paciente de 56 anos, posmenopausica, con un cancer de mama, que acude a la consulta oncologica, donde se evidencia, en el examen fisico, hirsutismo y otros elementos de virilizacion. Se aprecia elevacion marcada de la testosteronemia y otros androgenos. En la ecografia transvaginal y la tomografia computarizada de abdomenpelvis se observa una tumoracion anexial derecha solida. Se realiza histerectomia total mas anexectomia bilateral mas apendicectomia y omentectomia. En la anatomia patologica se evidencia un tumor de celulas de Leydig de variedad hiliar del ovario. Discusion: Los signos de virilizacion en mujeres posmenopausicas obedecen con mas frecuencia a trastornos de la funcion ovarica que adrenal. En el caso clinico presentado se diagnostico un tumor de celulas de Leydig de variedad hiliar, que se confirmo por la presencia caracteristica de cristales de Reinke. El tratamiento es quirurgico, y se recomienda la ooforectomia bilateral.

Three identical deoxyoligonucleotide third strands with a 3'-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cell beta-globin gene and the comparable wild-type sequence in linear duplex targets. Specificity and yield of UVA (365 nm) and visible (419 nm) light-induced photoadducts were studied. The total photoproduct yield varies with the linker and includes both monoadducts and crosslinks at various available pyrimidine sites. The specificity of photoadduct formation at the desired mutant T residue site was greatly improved by shortening the psoralen linker. In particular, using the N-4 linker, psoralen interaction with the residues of the non-coding duplex strand was essentially eliminated, while modification of the Sickle Cell mutant T residue was maximized. At the same time, the proportion of crosslink formation at the mutant T residue upon UV irradiation was much greater for the N-4 linker. The photoproducts formed with the wild-type target were fully consistent with its single base pair difference. The third strand with the N-4 linker was also shown to bind to a supercoiled plasmid containing the Sickle Cell mutation site, giving photoproduct yields comparable with those observed in the linear mutant target.

Analysis of extant proteomes has the potential of revealing how amino acid frequencies within proteins have evolved over biological time. Evidence is presented here that cysteine, tyrosine, and phenylalanine residues have substantially increased in frequency since the three primary lineages diverged more than three billion years ago. This inference was derived from a comparison of amino acid frequencies within conserved and non-conserved residues of a set of proteins dating to the last universal ancestor in the face of empirical knowledge of the relative mutability of these amino acids. The under-representation of these amino acids within last universal ancestor proteins relative to their modern descendants suggests their late introduction into the genetic code. Thus, it appears that extant ancient proteins contain evidence pertaining to early events in the formation of biological systems.

Eight base analogs were tested as third strand residues in otherwise homopyrimidine strands opposite each of the 'direct' (A.T and G.C) and 'inverted' (T.A and C.G) Watson-Crick base pairs, using UV melting profiles to assess triplex stability. The target duplexes contained 20 A.T base pairs and a central test base pair X.Y, while the third strand contained 20 T residues and a central Z test base. Z included 5-bromo-uracil, 5-propynyluracil, 5-propynylcytosine, 5-methyl-cytosine, 5-bromocytosine, hypoxanthine, 2-amino-purine and 2,6-diaminopurine. Some of the base analogs enhanced third strand binding to the target duplex with one or other 'inverted' central base pair relative to the binding afforded by any of the canonical bases. Other analogs did the same for the duplexes with the 'direct' target pairs. The increasing order of triplex stabilization by these base analogs is: opposite the 'inverted' base pairs, for T.A, A < C < 5-pC < 5-pU < T < 5-BrC < 5-meC < 5-BrU < 2-AP < 2,6-DAP < Hy < G, for C.G, 2-AP < A < Hy < G < 5-pC < 5-BrC < 5-meC < C < 2,6-DAP < T < 5-BrU < 5-pU; opposite the 'direct' base pairs, for A.T, 2-AP < A < 5-meC < C < G < Hy < 2,6-DAP < 5-pU < T = 5-BrU < 5-BrC < 5-pC, for G.C, G < 2,6-DAP < 2-AP < A < Hy < T < 5-BrU < 5-pU < 5-pC < 5-BrC < C < 5-meC.

A methodology has been developed for binding oligodeoxyribonucleotide 'third strands' to duplex DNA targets in fixed but not additionally denatured metaphase spreads and interphase nuclei under conditions found to be optimal in solution. Third-strand in situ hybridization (TISH) at pH 6.0 of a psoralen- and biotin-modified 16-nucleotide homopyrimidine third strand to a unique multicopy target sequence in human chromosome 17 alpha-satellite (D17Z1 locus) is described. UVA-photofixed third strands, rendered fluorescent by fluorescein isothiocyanate-labeled avidin, are reproducibly centromere-specific for chromosome 17, and visible without amplification of the signal in lymphocyte and somatic cell hybrid spreads and interphase nuclei. Two third-strand-specific D17Z1 haplotypes were identified. TISH has potential diagnostic, biochemical, and flow cytometric applicability to native metaphase and interphase chromatin.

A DNA third strand with a 3′-psoralen substituent was designed to form a triplex with the sequence downstream of the T·A mutant base pair of the human sickle cell β-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue “hook” complementary to an overhang of a linear duplex target was added to the 5′-end of the third strand via a T4 linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3′-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.

UV resonance Raman spectroscopy was used to detect and estimate the frequency of the unfavored imino tautomer of the transition mutagen 5-hydroxy-2′-deoxycytidine (HO 5 dCyt) in its anionic form. In DNA, this 2′-deoxycytidine analog arises from the oxidation of 2′-deoxycytidine and induces C → T transitions with 10 2 greater frequency than such spontaneous transitions. An imino tautomer marker carbonyl band (≈1650 cm −1 ) is enhanced at ≈65°C against an otherwise stable spectrum of bands associated with the favored amino tautomer. This band is similarly present in the UV resonance Raman spectra of the imino cytidine analogs N 3 -methylcytidine at high pH and N 4 -methoxy-2′-deoxycytidine at pH 7 and displays features attributable to the imino form of C residues and their derivatives. The fact that the imino tautomer of HO 5 dCyt occurs at a frequency consistent with its high mutagenic enhancement lends strong support to the hypothesis that unfavored base tautomers play important roles in the mispair intermediates of replication leading to substitution mutations.

3-Nitropyrrole (M) was introduced as a non-discriminating 'universal' base in nucleic acid duplexes by virtue of small size and a presumed tendency to stack but not hydrogen bond with canonical bases. However, the absence of thermally-induced hyperchromic changes by single-stranded deoxyoligomers in which M alternates with A or C residues shows that M does not stack strongly with A or C nearest neighbors. Yet, the insertion of a centrally located M opposite any canonical base in a duplex is sometimes even less destabilizing than that of some mismatches, and the variation in duplex stability is small. In triplexes, on the other hand, an M residue centrally located in the third strand reduces triplex stability drastically even when the X.Y target base pair is A.T or G. C in a homopurine. homopyrimidine segment. But, when the target duplex opposition is M-T and the third strand residue is T, the presence of M in the test triplet has little effect on triplex stability. Therefore, a lack of hydrogen bonding in an otherwise helix-compatible test triplet cannot be responsible for triplex destabilization when M is the third strand residue. Thus, M is non-discriminating and none-too-destabilizing in a duplex, but in a triplex it is extremely destabilizing when in the third strand.

The structure of the oligonucleotide d(A-G)10 in 0.6 M Na+, pH 7.0 has been investigated with UV resonance Raman (UVRR) spectroscopy. Variable wavelength excitation was used to distinguish the spectral contributions of dG and dA residues. Both classes of residues show UVRR hyperchromism with increasing temperature, reflecting unstacking of the bases. The dG residues melt relatively cooperatively with a Tm of approximately 42 degrees C. Unstacking is non-cooperative for the dA residues, increasing linearly between 4 and 80 degrees C. G-tetrads at low temperature are indicated by UVRR frequency shifts of modes associated with C6=O and C2-NH2 of the dG residues, and of vibrations involving N7, all sites of H-bonding. However, there are no indications of interbase H-bonds for the dA residues, showing they do not form H-bonded tetrads. Most of the bases are oriented anti about the glycosyl bond, but at 4 degrees C a fraction of the residues are syn. These results, together with the findings by Shiber et al. [Shiber,M.C., Braswell,E.H., Klump,H. and Fresco,J.R. (1996) Nucleic Acids Res. 24, 5004-5012] that d(A-G)10 under comparable conditions has the molecular weight of a dimer, support a model in which two hairpins interact to form a helical structure with G-tetrads and intercalated dA residues.

Little has been done to study the effectiveness of antidepressants in controlling anxiety/depression in a population of cancer patients. A double-blind placebo-controlled study was therefore designed to assess the effectiveness of 20 mg fluoxetine. Of 115 cancer patients who fulfilled entry criteria for levels of distress, 45 patients were randomized to a fluoxetine treatment group (FA) and 46 patients to a placebo group (PA) after a 1-week placebo period designed to exclude placebo responders. The Montgomery and Asberg Depression Scale (MADRS), the Hamilton Anxiety Scale (HAS), the Hospital Anxiety and Depression Scale (HADS), the Revised Symptom Checklist (SCL90-R) and the Spitzer Quality of Life Index (SQOLI) were used to assess the efficacy of fluoxetine. The response rate, defined by a HADS score lower than 8 after 5 weeks of treatment, was not significantly higher in the FA group (11%) compared to the PA group (7%). Compared to the PA group, patients in the FA group showed a significantly greater decrease in SCL90-R mean total score after 5 weeks, but not a greater decrease in HADS mean score. No difference between the two groups was found in observer-reported assessments (MADRS, HAS and SQOLI). Significantly more drop-outs were observed in the FA group (n = 15) than in the PA group (n = 7), although the frequencies of side-effects were not significantly different.

UV resonance Raman spectroscopy has been utilized to directly observe structural features of the recently described nucleic acid single-stranded helix d(A(+)-G)10. An absence of base stacking is confirmed by invariant hypochromic ratios of dominant vibrational modes for the oligomer relative to its constituent monomers as the structure is thermally denatured. The N1 of dA residues is protonated, as determined by similarity to the ring-stretching vibrations for protonated adenine and its derivatives. Selective resonance enhancement of Raman vibrational modes from dA and dG residues shows frequency shifts upon thermal denaturation that confirm the participation of the exocyclic amino of dA but not dG residues in H-bonding. Conformationally sensitive glycosyl bond modes suggest anti residue conformations.

A novel nucleic acid secondary structure, exemplified by d(A+-G)lo, is formed by an intramolecular, cooperative, acid-induced, coil - helix transition. The helix is apparently left-handed, lacks base stacking and pairing, and is maintained by hydrogen and ionic bonds between dA+ "side- chain" residues (with electropositive hydrogens -N6H2, -N,+H) and the phosphodiester backbone. Modeling indicates that those dA+ residues lie approximately parallel to the helix axis, interacting with the n - 1 backbone phosphates (with electronegative oxygens), somewhat like the -C=O-H-N- longitudinal interactions in a protein a-helix. Moreover, the intervening dG side-chain residues are extrahelical, as are amino acid side chains of an a-helix. All currently recognized types of nucleic acid secondary structure involve helical stacks of bases. Such stacks occur in single strands, in base pairs of hairpins formed by single strands, in base pairs of two-stranded complexes, in base triplets of three-stranded structures, and in base quartets of four-stranded complexes. In this paper, we provide details of an entirely different type of nucleic acid helical secondary structure, one that does not depend for its stabilization on helically wound stacks of bases or base pairs. Rather, this novel type of secondary structure encompasses single strands of the repeating ho- mopurine doublet sequence d(A-G),, helically twisted by an unusual combination of ionic and hydrogen (H) bonds essentially parallel to the helix axis. We first came upon this nucleic acid secondary structure in the course of investigating the molecular properties of d(A- G)lo, which displays an intense differential absorption of circularly polarized light (CD) without concomitant develop- ment of hypochromism in the near-ultraviolet (UV) when the pH is reduced from neutrality. Additional observations enabled attribution of this unusual dichotomy in optical properties to a single-stranded helical secondary structure stabilized largely by Coulombic interactions between nega- tively charged backbone phosphates and distal protonated dA (dA+) residues that do not overlap with their nearest neighbor dG residues. Although these properties made it possible to deduce some regular features of the secondary structure of d(A+-G)lo, direct evidence for many structural ~

A major variety of "spontaneous" genomic damage is endogenous generation of apurinic sites. Depurination rates vary widely across genomes, occurring with higher frequency at "depurination hot spots." Recently, we discovered a site-specific self-catalyzed depurinating activity in short (14-18 nucleotides) DNA stem-loop-forming sequences with a 5'-G(T/A)GG-3' loop and T·A or G·C as the first base pair at the base of the loop; the 5'-G residue of the loop self-depurinates at least 10(5)-fold faster than random "spontaneous" depurination at pH 5. Formation of the catalytic intermediate for self-depurination in double-stranded DNA requires a stem-loop to extrude as part of a cruciform. In this study, evidence is presented for self-catalyzed depurination mediated by cruciform formation in plasmid DNA in vitro. Cruciform extrusion was confirmed, and its extent was quantitated by digestion of the plasmid with single strand-specific mung bean endonuclease, followed by restriction digestion and sequencing of resulting mung bean-generated fragments. Appearance of the apurinic site in the self-depurinating stem-loop was confirmed by digestion of plasmid DNA with apurinic endonuclease IV, followed by primer extension and/or PCR amplification to detect the endonuclease-generated strand break and identify its location. Self-catalyzed depurination was contingent on the plasmid being supercoiled and was not observed in linearized plasmids, consistent with the presence of the extruded cruciform in the supercoiled plasmid and not in the linear one. These results indicate that self-catalyzed depurination is not unique to single-stranded DNA; rather, it can occur in stem-loop structures extruding from double-stranded DNA and therefore could, in principle, occur in vivo.

This chapter focuses on the nature of site-specific self-catalyzed DNA depurination as a spontaneous mechanism inherent in the chemical structure and dynamics of DNA that has contributed to evolutionary change. It describes the essential molecular features of the mechanism, the short consensus sequence elements that form the catalytic intermediate, the basics of the reactions that lead to the creation of apurinic sites, and the means by which those sites give rise to substitution and short deletion mutations. The consensus sequences are widely distributed in double-stranded genomes across the phyla at high frequency that increases up the phylogenetic tree. In the human genome, they constitute >2 × 106 potential mutagenic sites, non-randomly scattered among very many genes, some containing multiple sites. Examples are presented of genes in which the mutations coincide with their self-depurination consensus sequences, the most striking being those in the β-globin gene that are responsible for six anemias and two β-thalassemias. Those of the olfactory receptor genes and the hypervariable regions of the immunoglobulin genes are shown to have utilized the mechanism to evolve their high degree of diversity and/or to develop their contemporaneous diversity for their present function.

This work was supported by National In- stitutes of Health Grants GM 25158 (to T.G.S.) and GM 42936 (to J.R.F.) and by National Science Foundation Grant DMB 84 19060 (to J.R.F.).

The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.

Introduction L’echographie pleuro-pulmonaire (EP) et la radiographie pulmonaire (RP) sont recommandees dans la prise en charge du traumatise grave. La puissance diagnostique de l’echographie est superieure a celle de la radiographie pour les epanchements pleuraux. L’impact therapeutique des deux techniques en association est peu etudie. L’objectif principal etait de comparer la puissance diagnostique de l’EP et de la RP pour le diagnostic du pneumothorax (PNO) et de l’hemothorax. L’objectif secondaire etait d’evaluer l’impact therapeutique respectif de ces procedures. Materiel et methodes Etude retrospective monocentrique (CHU Nord, Marseille) avec collecte prospective des donnees. Pour chaque patient traumatise primaire, une RP, une EP et une TDM corps entier etaient realisees. Selon les protocoles de service, une fiche de recueil des resultats des imageries et des traitements etait remplie avec une analyse retrospective. Les dossiers des patients etaient ensuite relus (un seul relecteur) pour determiner si les decisions etaient appropriees. La puissance diagnostique de la RP et de l’EP etaient definies par rapport a la TDM. L’impact therapeutique etait defini par le nombre de modifications induites par les examens d’imagerie (RP, EP ou RP + EP) avant TDM rapportes au nombre d’examens realises. Resultats En 30 mois, 146 patients traumatises primaires ont ete inclus dans l’etude. Les puissances diagnostiques et impacts therapeutiques des deux examens sont resumes dans le Tableau 1 . En cas d’EP anormale, l’impact therapeutique de la RP etait de 28 %. En cas d’EP normale, il etait nul. Les PNO n’etaient draines que sur une positivite des deux examens (EP + RP). Trois hemothorax non detectes a la RP ont ete draines sur les donnees de l’EP. Aucun drainage inapproprie n’etait detecte a la relecture des dossiers. Trois abstentions de drainage ont ete analyses comme inappropriees (PNO draines secondairement non detectes a l’EP et a la RP). Discussion L’EP a une puissance diagnostique et un impact therapeutique superieurs a la RP sans toutefois etre parfaite dans le contexte de l’urgence. L’association des deux techniques evite de drainer a tort des PNO minimes detectes par l’EP. Cependant, si l’EP est normale, la RP n’apportait aucune modification therapeutique. Les consequences d’une prescription de RP basee sur les resultats de l’EP en termes de qualite de prise en charge initiale restent a evaluer par de futures etudes.

We have identified a type of secondary structure for the homopurine oligomer d(A-G)10 below pH 6 in 0.01 M Na+ that is characterized by intense CD but only minor hypochromicity. The stability of this helix, designated d(A(+)-G)10, does not depend on oligomer concentration and increases sharply as ionic strength or pH drops, reaching a maximum at 4.0 (melting temperature, 37 degrees C). The pKa for the transition, 5.3 at 25 degrees C and even higher with decreasing temperature and [Na+], is much higher than the intrinsic pKa values for dA or dG residues. While the dA residues are protonated in the helix, further protonation of the dG residues disrupts it. When observed at 280 nm, melting of the helix first results in hypochromicity due to stacking of extrahelical dG residues with neighboring dA residues. The character and temperature dependence of the CD spectra of the constituent dinucleoside monophosphates indicate minimal chirality and base overlap for the A+pG sequences in d(A(+)-G)10 but left-handed twist with some base overlap for the GpA+ sequences. The observed properties are best satisfied by a model for an intramolecular helix with limited base overlap, stabilized by ionic bonds between dA residues protonated at N-1 and downstream negatively charged phosphates brought close due to the backbone helical twist, while Gsyn residues lie external to the helix. This structure could provide additional stabilizing energy for biologically relevant protonated non-B-DNA structures adopted by homopurine.homopyrimidine sequences due to topological stress or specific protein binding.

A self-catalyzed, site-specific guanine-depurination activity has been found to occur in short gene sequences with a potential to form a stem-loop structure. The critical features of that catalytic intermediate are a 5′-G-T-G-G-3′ loop and an adjacent 5′-T·A-3′ base pair of a short duplex stem stable enough to fix the loop structure required for depurination of its 5′-G residue. That residue is uniquely depurinated with a rate some 5 orders of magnitude faster than that of random “spontaneous” depurination. In contrast, all other purine residues in the sequence depurinate at the spontaneous background rate. The reaction requires no divalent cations or other cofactors and occurs under essentially physiological conditions. Such stem-loops can form in duplex DNA under superhelical stress, and their critical sequence features have been found at numerous sites in the human genome. Self-catalyzed stem-loop-mediated depurination leading to flexible apurinic sites may therefore serve some important biological role, e.g., in nucleosome positioning, genetic recombination, or chromosome superfolding.

The precise mechanism of stop codon recognition in translation termination is still unclear. A previously published study by Ivanov and colleagues proposed a new model for stop codon recognition in which 3-nucleotide Ter-anticodons within the loops of hairpin helices 69 (domain IV) and 89 (domain V) in large ribosomal subunit (LSU) rRNA recognize stop codons to terminate protein translation in eubacteria and certain organelles. We evaluated this model by extensive bioinformatic analysis of stop codons and their putative corresponding Ter-anticodons across a much wider range of species, and found many cases for which it cannot explain the stop codon usage without requiring the involvement of one or more of the eight possible noncomplementary base pairs. Involvement of such base pairs may not be structurally or thermodynamically damaging to the model. However, if, according to the model, Ter-anticodon interaction with stop codons occurs within the ribosomal A-site, the structural stringency which that site imposes on sense codon·tRNA anticodon interaction should also extend to stop codon·Ter-anticodon interactions. Moreover, with Ter-tRNA in place of an aminoacyl-tRNA, for each of the various Ter-anticodons there is a sense codon that can interact with it preferentially by complementary and wobble base-pairing. Both these considerations considerably weaken the arguments put forth previously.

The thermal stability of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6) and d(T)(21):d(A)(21);d(T)(21) was studied in the presence of high concentrations of the anions Cl(-), HPO(4)(2-), CH(3)COO(-), SO(4)(2-) and ClO(4)(-). Thermally-induced triplex and duplex transitions were identified by UV- and CD-spectroscopy and T(m) values were determined from melting profiles. A thermodynamic analysis of triplex transitions shows the limitations of commonly used treatments for determining the associated release or uptake of salt, solute or water. Enhancement of the stability of these triplexes follows the rank order of the Hofmeister series for anions of sodium and ammonium salts, whereas water structure-breaking solutes have the opposite effect. The rank order for the Hofmeister series ClO(4)(-)

The overall goal was to explore whether nucleic acid third strands could be used to bind with very high specificity to specific targets within whole genomes. Towards this end conditions had to be found to keep erroneous binding to an absolute minimum. The goal to use third strands (linked to magnetic beads) to ''capture'' large particles such as plasmids, cosmids, and whole chromosomes from complex mixtures was partially met; their use to serve as cytogenetic probes of metaphase chromosomes and to deliver reactive reagents to unique target sites on chromosomes in vivo for the purpose of mutagenizing specific base pairs was fully met; and their use as cytogenetic probes of chromosomal DNA in sections of formalin-fixed, paraffin-embedded tissue has been met since the DOE support was terminated.

The possibility that RNY pattern bias in extant sequences is a remnant of more pronounced bias of this type in early ancestors was investigated. To this end, conserved residues (those residues for which the inferred ancestral and known descendant amino acids are identical) and non-conserved residues of ancient proteins dating to the Last Universal Ancestor were identified within six species: two archaea, two eubacteria and two eukaryotes. Bias within sequence elements encoding each subset of residues, conserved and non-conserved, was then determined. In all species, GNN bias is greater within conserved than non-conserved sequence elements, whereas ANN is not. This difference is statistically significant in all six species examined. Since the relative mutability of the GNN-encoded amino acids does not explain the greater bias in conserved sequences, it is concluded that early sequences probably possessed a strong GNN bias. It is suggested that this bias may be a consequence of the GNN codons being the first introduced into the genetic code. Although NNY bias is also greater within conserved sequence elements of the six species, that difference is statistically significant in only half of them. Therefore, the evidence for early NNY bias remains inconclusive. The findings of this study do not support the proposal of Diaz-Lazcoz et al. (J. Mol. Biol. 250 (1995) 123) that the codons of the TCN four-codon block were the first assigned to serine during the evolution of the genetic code.

Publisher Summary This chapter describes and rationalizes the several equilibria that underlie the conformational polymorphism of d(A-G) n . It describes each of the equilibria along with the properties of their participating conformers. Additionally, as various of these d(A-G) n conformations seem to share the physical–chemical properties with naturally occurring irregular, nonrepetitive, homopurine sequences in both DNA and RNA, the chapter presents the use of more readily interpretable properties of the regular d(A-G) n repeat to extrapolate structure for those irregular sequences and vice versa. The chapter describes five discrete conformations undertaken by the sequence d(A-G) n between n=6 and 30, and the equilibria between the different conformers. It is noted that the knowledge gained from studies of the multiple conformations available to d(A-G) n sequences such as those occurring in strand segments of one or the other complementary strands of DNA, along with the indications of their biological significance, should pave the way for the possibility of other repetitive strand elements whose conformational potential may have other functional consequences.

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.

A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3′-untranslated region (3′-UTR) and inserted into 3′-UTR or 3′ flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5′ flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5′ and 3′ flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3′-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.

A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.

In contrast to shorter homologs which only form a single-stranded nucleic acid alpha-helix in acid solution at [Na+]/=0.2 M) the protonated base-backbone interactions are so weakened that duplex stability becomes increasingly dependent upon H-bonded base pairing and stacking and almost independent of pH. Between pH 6 and 8 this duplex structure is devoid of protonated dA residues and shows positive dependence of T m on ionic strength similar to that of DNA.

d(A-G)10 forms two helical structures at neutrality, at low ionic strength a single-hairpin duplex, and at higher ionic strength a double-hairpin tetraplex. An ionic strength-dependent equilibrium between these forms is indicated by native PAGE, which also reveals additional single-stranded species below 0.3 M Na+, probably corresponding to partially denatured states. The equilibrium also depends upon oligomer concentration: at very low concentrations, d(A-G)10 migrates faster than the random coil d(C-T)10, probably because it is a more compact single hairpin; at high concentrations, it co-migrates with the linear duplex d(A-G)10 x d(C-T)10, probably because it is a two-hairpin tetraplex. Molecular weights measured by equilibrium sedimentation in 0.1 M Na+, pH 7, reveal a mixture of monomer and dimer species at 1 degree C, but only a monomer at 40 degrees C; in 0.6 M Na+, pH 7, only a dimer species is observed at 4 degrees C. That the single- and double-stranded species are hairpin helices, is indicated by preferential S1 nuclease cleavage at the center of the oligomer(s), i.e., the loop of the hairpin(s). The UV melting transition below 0.3 M Na+ or K+, exhibits a dTm/dlog[Na+/K+] of 33 or 36 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex to a single-hairpin duplex with extrahelical residues. When [Na+/K+] > or = 0.3 M, dTm/dlog [Na+/K+] is 19 or 17 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex directly to single strands. A two-hairpin structure stabilized by G-tetrads is indicated by differential scanning calorimetry in 0.15 M Na+/5 mM Mg2+, with deltaH of formation per mole of the two-hairpin tetraplex of -116.9 kcal or -29.2 kcal/mol of G-tetrad.

Near-UV difference spectral analysis of the triplex formed from d(C-T)6 and d(A-G)6.d(C-T)6 in neutral and acidic solution shows that the third strand dC residues are protonated at pH 7.0, far above their intrinsic pKa. Additional support for ion-dipole interactions between the third strand dC residues and the G.C target base pairs comes from reduced positive dependence of triplet stability on ionic strength below 0.9 M Na+, inverse dependence above 0.9 M Na+ and strong positive dependence on hydrogen ion concentration. Molecular modeling (AMBER) of C:G.C and C+:G.C base triplets with the third strand base bound in the Hoogsteen geometry shows that only the C+:G.C triplet is energetically feasible. van't Hoff analysis of the melting of the triplex and target duplex shows that between pH 5.0 and 8.5 in 0.15 M NaCl/0.005 M MgCl2 the enthalpy of melting (delta H degree obs) varies from 5.7 to 6.6 kcal.mol-1 for the duplex in a duplex mixture and from 7.3 to 9.7 kcal.mol-1 for third strand dissociation in the triplex mixture. We have extended the condensation-screening theory of Manning to pH-dependent third strand binding. In this development we explicitly include the H+ contribution to the electrostatic free energy and obtain [formula: see text]. The number of protons released in the dissociation of the third strand from the target duplex at pH 7.0, delta n2, is thereby calculated to be 5.5, in good agreement with approximately six third strand dc residues per mole of triplex. This work shows that when third strand binding requires protonated residues that would otherwise be neutral, triplex formation and dissociation are mediated by proton uptake and release, i.e., a proton switch. As a by-product of this study, we have found that at low pH the Watson-Crick duplex d(A-G)6.d(C-T)6 undergoes a transition to a parallel Hoogsteen duplex d(A-G)6.d(C(+)-T)6.

A double emulsion, film dehydration/rehydration approach was developed for encapsulation of hemoglobin (Hb) at high concentration in liposomes. The liposome-encapsulated Hb (LEH) membrane was formulated to contain either phosphatidylinositol (PI) or polyethyleneglycol phosphatidylethanolamine (PEG-PE) along with partially hydrogenated egg-PC, cholesterol, and α-tocopherol in a molar ratio of 0.1:1:1:0.02, respectively. The methods introduced in this study followed a multi-step procedure. First, a primary emulsion of Hb in organic solvent containing dissolved lipids was formed. Next, the emulsion was dispersed into an aqueous continuous phase to form a water-in-oil-in-water type double emulsion. Other than the lipids noted above, no surfactants were used in this system. The double emulsion was then converted to LEH by the following steps: evaporating the organic solvent; dehydrating the water to form a dry, thin Hb-lipid film; rehydrating the film in Hb solution to form the LEH; reducing the size of the LEH using ‘microfluidization’ i.e., high pressure/hydrodynamic shear; and lastly washing the down-sized LEH in buffer. Physico-chemical properties of the model LEH were measured, including oxygen content, encapsulated Hb concentration, oxygen affinity and cooperativity, vesicular size distribution, viscosity, and stability. The suitability of LEH prepared in this manner as a red blood cell substitute was shown using continuous isovolemic exchange transfusion techniques in a small animal model: clearance, efficacy and acute toxicity were evaluated.

22203 Background: HNPCC is the most common colorectal cancer syndrome and is associated with germ-line mutations in DNA mismatch repair genes (MMR). To date, more than 700 MMR gene mutations have b...

3730 Background: Non-polypotic familiar colon cancer (HNPCC) is an autosomical dominant transmitted syndrome with high aggregation of colorectal cancer (CRC), frequently associated with other solid tumors. Few studies have investigated the frequency of HNPCC in patients (pts.) having undergone surgery for CRC. These have shown a marked geographic variation, ranging from 0.5% to 13%. The aim of this study is to estimate the frequency of HNPCC in the Uruguayan population. Methods: We performed a retrospective analysis of a cohort of non-selected pts. operated for CRC in the Hospital Central de las Fuerzas Armadas (Montevideo, Uruguay) between January 1987 and December 2002. Cases were classified in 3 groups according to personal and family history: 1) those fulfilling Amsterdam criteria, classified as HNPCC; 2) those not fulfilling Amsterdam criteria but considered as a population of increased risk for cancer (PIR); and 3) those classified as sporadic CRC. Genetic analysis to detect mutations in hMLH1 and h...

Tm values of 16 fully complementary RNA duplexes with repeating base sequence have been employed as the empirical basis for developing a reliable and practical method for computing apparent enthalpies (ΔHcalc) for their helix → coil transitions. The approach taken is the same as in the accompanying investigation of DNA duplexes, although some of the computational variables of the “best-fit” function are necessarily different due to the distinguishing structural properties of the RNA-type helix. An excellent linear correlation was thus obtained between experimental Tm and ΔHcalc values. An equally good fit was obtained between Tm and ΔHcalc for five unrelated (to the 16 RNAs) decaribonucleotide duplexes. The differences in computational variables between the best-fit methods for RNA and DNA duplexes are shown to be a reflection of differences in cation binding and the effective local dielectric. The greater Tm dependence on G·C content of RNA helices than of DNA helices is shown to be due to a greater latitude of stacking stabilities of complementary dinucleotide fragments containing A·T than A·U base pairs.