Alumorphs--human DNA polymorphisms detected by polymerase chain reaction using Alu-specific primers

The simultaneous analysis of multiple loci could substantially increase the efficiency of mapping studies. Toward this goal, we used the polymerase chain reaction to amplify multiple DNA fragments originating from dispersed genomic segments that are flanked by Alu repeats. Analysis of different human DNA samples revealed numerous amplification products distinguishable by size, some of which vary between individuals. A family study demonstrated that these polymorphic fragments are inherited in a Mendelian fashion. Because of the ubiquitous distribution of Alu repeats, these markers, called "alumorphs," could be useful for linkage mapping of the human genome. A major advantage of alumorphs is that no prior knowledge of DNA sequence of marker loci is required. This approach may find general application for any genome where interspersed repetitive sequences are found.     

Cooperativity in low-affinity Mg2+ binding to tRNA

The Pb2+-catalyzed cleavage of tRNAPhe has been used to probe the effect of Na+ and Mg2+ binding to tRNA. Na+ is a noncompetitive inhibitor of the Pb2+-catalyzed cleavage. Millimolar Mg2+ is also a noncompetitive inhibitor. Analysis of the Mg2+ data show that at least two sites are involved in binding and that there is an interaction between the sites (cooperativity). Low-affinity Mg2+ binding is thus different from "weak" and "strong" Mg2+ binding to tRNA characterized previously. We postulate that the alterations induced by low-affinity Mg2+ binding in tRNA mimic to some extent those brought about in RNA by the interaction with a protein factor and that at appropriate [Mg2+] the whole structure of tRNA is able to respond in a concerted way to a signal from the environment such as aminoacylation or codon binding.     

Mechanism of codon recognition by transfer RNA and codon-induced tRNA association

The steps of UUC recognition by tRNAPhe were analysed by temperature-jump measurements. At ion concentrations close to physiological conditions we found three relaxation processes, which we assigned to (1) formation of codon-anticodon complexes, (2) a conformational change of the anticodon loop coupled with Mg2+ binding, and (3) codon-induced association of tRNA. The relaxation data were evaluated both by the usual procedure (fitting the exponentials evaluated from the individual experiments of a set to a reaction model) and by "global fitting", i.e. fitting a set of relaxation curves obtained at various concentrations directly to a reaction model, thus leaving out the intermediate exponential fitting step. The data can be represented quantitatively by a three-step model: the codon binds to the anticodon at a rate of 4 X 10(6) to 6 X 10(6) M-1S-1 as is usual for the formation of oligomer helices; the conformation change of the anticodon loop is associated with inner sphere complexation of Mg2+ at a rate of 10(3) S-1; the codon-tRNA complexes form dimers at a rate of 5 X 10(6) to 15 X 10(6) M-1S-1. A similar mechanism is found for the binding of the wobble codon UUU to tRNAPhe at increased concentrations of Mg2+. Measurements at different Mg2+ concentrations demonstrate the distinct role of this ion in the codon recognition and the codon-induced tRNA dimerization. We propose a simple mechanism, based upon the special properties of magnesium ions, for long-distance transfer of reaction signals along nucleic acid chains.     

Overall Informativity,OI, in DNA Polymorphisms Revealed by inter-AluPCR: Detection of Genomic Rearrangements

We studied two systems of multilocus markers revealed by PCR using primers directing amplification betweenAlurepeats in a tail-to-tail orientation. Genomic polymorphisms were detected as the presence or absence of the electrophoretic bands representing DNA fragments of a given length. A total of 104 such fragments segregating as Mendelian markers in a panel of eight CEPH families were analyzed by two-point linkage analysis. Fifty-one of these fragments were localized with respect to CEPH markers; they represented 33 loci, 7 of which were multiallelic. Locus-specific oligonucleotides were developed and used as hybridization probes to identify the mapped loci within a complex pattern of inter-AluPCR products. A great proportion of inter-AluPCR polymorphisms represented length variants within amplified DNA segments, while others were presumably due to mutations within the priming sites. To describe the expected number of informative loci per typing experiment we introduced a parameter called overall informativity (OI), which provides a single measure of the multiplex ratio and the informativity of markers contributing to a multilocus system (OIof a single locus is equivalent to its heterozygosity and cannot exceed 0.5 for a biallelic codominant marker). HighOIvalues (5.8 and 11.5) of the two presented systems of inter-AluPCR markers of random chromosomal distribution render them suitable for mapping genomic rearrangements such as genomic deletions in tumoral tissues. This was illustrated by the detection of loss of heterozygosity in the 9q22–qter region in sporadic colon cancer.     

Evolution of secondary structure in the family of 7SL-like RNAs

Primate and rodent genomes are populated with hundreds of thousands copies of Alu and B1 elements dispersed by retroposition, i.e., by genomic reintegration of their reverse transcribed RNAs. These, as well as primate BC200 and rodent 4.5S RNAs, are ancestrally related to the terminal portions of 7SL RNA sequence. The secondary structure of 7SL RNA (an integral component of the signal recognition particle) is conserved from prokaryotes to distant eukaryotic species. Yet only in primates and rodents did this molecule give rise to retroposing Alu and B1 RNAs and to apparently functional BC200 and 4.5S RNAs. To understand this transition and the underlying molecular events, we examined, by comparative analysis, the evolution of RNA structure in this family of molecules derived from 7SL RNA.RNA sequences of different simian (mostly human) and prosimian Alu subfamilies as well as rodent B1 repeats were derived from their genomic consensus sequences taken from the literature and our unpublished results (prosimian and New World Monkey). RNA secondary structures were determined by enzymatic studies (new data on 4.5S RNA are presented) and/or energy minimization analyses followed by phylogenetic comparison. Although, with the exception of 4.5S RNA, all 7SL-derived RNA species maintain the cruciform structure of their progenitor, the details of 7SL RNA folding domains are modified to a different extent in various RNA groups. Novel motifs found in retropositionally active RNAs are conserved among Alu and B1 subfamilies in different genomes. In RNAs that do not proliferate by retroposition these motifs are modified further. This indicates structural adaptation of 7SL-like RNA molecules to novel functions, presumably mediated by specific interactions with proteins; these functions were either useful for the host or served the selfish propagation of RNA templates within the host genome.Abbreviations FAM fossil Alu element - FLAM free left Alu monomer - FRAM free right Alu monomer - L-Alu left Alu subunit - R-Alu right Alu subunit Correspondence to: D. LabudaDedicated to Dr. Robert Cedergren on the occasion of his 25th anniversary at the University of Montreal     

Ubiquitous mammalian-wide interspersed repeats (MIRs) are molecular fossils from the mesozoic era.

Short interspersed elements (SINEs) are ubiquitous in mammalian genomes. Remarkable variety of these repeats among placental orders indicates that most of them amplified in each lineage independently, following mammalian radiation. Here, we present an ancient family of repeats, whose sequence divergence and common occurrence among placental mammals, marsupials and monotremes indicate their amplification during the Mesozoic era. They are called MIRs for abundant Mammalian-wide Interspersed Repeats. With approximately 120,000 copies still detectable in the human genome (0.2-0.3% DNA), MIRs represent a 'fossilized' record of a major genetic event preceding the radiation of placental orders.     

Superoxide dismutase mimetic activity of a cyclic tetrameric Schiff base N-coordinated Cu(II) complex

A pulse radiolytic study using the cyclic tetrameric Schiff base N-coordinated copper complex Cu(TAAB)²⁺ has been performed. The reaction of the Cu(TAAB)²⁺ complex with superoxide revealed pseudo first-order characteristics with the rate constant of k ₂ = (2.9 ± 0.5) × 10⁸ mol^–1 s^–1 dm³. The complex survive presence of competing serum albumin in physiological concentrations. The complex stability constant K = 1.15 × 10¹⁸ (log K = 18.06) is two orders of magnitude higher than that of Cu(II)-serum albumin (log K = 16.2). Transient changes of the stability during the oxidation/reduction process and in the presence of 600 /mol l^–1 albumin did not affect significantly either the electronic absorption of the complex or its catalytic activity.