Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers.

Urine samples from 20 male volunteers of European Caucasian origin were stored at 4 degrees C over a 4-month period in order to compare the identification potential of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) markers. The amount of nDNA recovered from urines dramatically declined over time. Consequently, nDNA likelihood ratios (LRs) greater than 1,000 were obtained for 100, 70 and 55% of the urines analysed after 6, 60 and 120 days, respectively. For the mtDNA, HVI and HVII sequences were obtained for all samples tested, whatever the period considered. Nevertheless, the highest mtDNA LR of 435 was relatively low compared to its nDNA equivalent. Indeed, LRs obtained with only three nDNA loci could easily exceed this value and are quite easier to obtain. Overall, the joint use of nDNA and mtDNA markers enabled the 20 urine samples to be identified, even after the 4-month period.

Transposase and cointegrase: specialized transposition proteins of the bacterial insertion sequence IS21 and related elements.

The bacterial insertion sequence IS21 shares with many insertion sequences a two-step, reactive junction transposition pathway, for which a model is presented in this review: a reactive junction with abutted inverted repeats is first formed and subsequently integrated into the target DNA. The reactive junction occurs in IS21-IS21 tandems and IS21 minicircles. In addition, IS21 shows a unique specialization of transposition functions. By alternative translation initiation, the transposase gene codes for two products: the transposase, capable of promoting both steps of the reactive junction pathway, and the cointegrase, which only promotes the integration of reactive junctions but with higher efficiency. This review also includes a survey of the IS21 family and speculates on the possibility that other members present a similar transpositional specialization.

Nonshivering thermogenesis capacity associated to mitochondrial DNA haplotypes and gender in the greater white-toothed shrew, Crocidura russula.

A selection gradient was recently suggested as one possible cause for a clinal distribution of mitochondrial DNA (mtDNA) haplotypes along an altitudinal transect in the greater white-toothed shrew, Crocidura russula (Ehinger et al. 2002). One mtDNA haplotype (H1) rare in lowland, became widespread when approaching the altitudinal margin of the distribution. As H1 differs from the main lowland haplotype by several nonsynonymous mutations (including on ATP6), and as mitochondria play a crucial role in metabolism and thermogenesis, distribution patterns might stem from differences in the thermogenic capacity of different mtDNA haplotypes. In order to test this hypothesis, we measured the nonshivering thermogenesis (NST) associated with different mtDNA haplotypes. Sixty-two shrews, half of which had the H1 haplotype, were acclimated in November at semioutdoor conditions and measured for NST throughout winter. Our results showed the crucial role of NST for winter survival in C. russula. The individuals that survived winter displayed a higher significant increase in NST during acclimation, associated with a significant gain in body mass, presumably from brown fat accumulation. The NST capacity (ratio of NST to basal metabolic rate) was exceptionally high for such a small species. NST was significantly affected by a gender x haplotype interaction after winter-acclimation: females bearing the H1 haplotype displayed a better thermogenesis at the onset of the breeding season, while the reverse was true for males. Altogether, our results suggest a sexually antagonistic cyto-nuclear selection on thermogenesis.

Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA.

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5'-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.

Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I.

Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.

ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

A user's guide to the encyclopedia of DNA elements (ENCODE).

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome.

Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments.

The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.

Molecular characterization of HLA-C incompatibilities in HLA-ABDR-matched unrelated bone marrow donor-recipient pairs. Sequence of two new Cw alleles (Cw*02023 and Cw*0707) and recognition by cytotoxic T lymphocytes.

While the influence of HLA-AB and -DRB1 matching on the outcome of bone marrow transplantation (BMT) with unrelated donors is clear, the evaluation of HLA-C has been hampered by its poor serological definition. Because the low resolution of standard HLA-C typing could explain the significant number of positive cytotoxic T lymphocyte precursor frequency (CTLpf) tests found among HLA-AB-subtype, DRB1/B3/B5-subtype matched patient/donor pairs, we have identified by sequencing the incompatibilities recognized by CD8+ CTL clones obtained from such positive CTLpf tests. In most cases the target molecules were HLA-C antigens that had escaped detection by serology (e.g. Cw*1601, 1502 or 0702). Direct recognition of HLA-C by a CTL clone was demonstrated by lysis of the HLA class I-negative 721.221 cell line transfected with Cw*1601 cDNA. Because of the functional importance of Cw polymorphism, a PCR-SSO oligotyping procedure was set up allowing the resolution of 29 Cw alleles. Oligotyping of a panel of 382 individuals (including 101 patients and their 272 potential unrelated donors, 5 related donors and 4 platelet donors) allowed to determine HLA-C and HLA A-B-Cw-DRB1 allelic frequencies, as well as a number of A-Cw, B-Cw, and DRB1-Cw associations. Two new HLA-Cw alleles (Cw*02023 and Cw*0707) were identified by DNA sequencing of PCR-amplified exon 2-intron 2-exon 3 amplicons. Furthermore, we determined the degree of HLA-C compatibility in 287 matched pairs that could be formed from 73 patients and their 184 potential unrelated donors compatible for HLA-AB by serology and for HLA-DRB1/ B3/B5 by oligotyping. Cw mismatches were identified in 42.1% of these pairs, and AB-subtype oligotyping showed that 30% of these Cw-incompatible pairs were also mismatched for A or B-locus subtype. The degree of HLA-C incompatibility was strongly influenced by the linkage with B alleles and by the ABDR haplotypes. Cw alleles linked with B*4403, B*5101, B18, and B62 haplotypes were frequently mismatched. Apparently high resolution DNA typing for HLA-AB does not result in full matching at locus C. Since HLA-C polymorphism is recognized by alloreactive CTLs, such incompatibilities might be as relevant as AB-subtype mismatches in clinical transplantation.

Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes.

BACKGROUND: The exceptionally diverse species flocks of cichlid fishes in East Africa are prime examples of parallel adaptive radiations. About 80% of East Africa's more than 1 800 endemic cichlid species, and all species of the flocks of Lakes Victoria and Malawi, belong to a particularly rapidly evolving lineage, the haplochromines. One characteristic feature of the haplochromines is their possession of egg-dummies on the males' anal fins. These egg-spots mimic real eggs and play an important role in the mating system of these maternal mouthbrooding fish. RESULTS: Here, we show that the egg-spots of haplochromines are made up of yellow pigment cells, xanthophores, and that a gene coding for a type III receptor tyrosine kinase, colony-stimulating factor 1 receptor a (csf1ra), is expressed in egg-spot tissue. Molecular evolutionary analyses reveal that the extracellular ligand-binding and receptor-interacting domain of csf1ra underwent adaptive sequence evolution in the ancestral lineage of the haplochromines, coinciding with the emergence of egg-dummies. We also find that csf1ra is expressed in the egg-dummies of a distantly related cichlid species, the ectodine cichlid Ophthalmotilapia ventralis, in which markings with similar functions evolved on the pelvic fin in convergence to those of the haplochromines. CONCLUSION: We conclude that modifications of existing signal transduction mechanisms might have evolved in the haplochromine lineage in association with the origination of anal fin egg-dummies. That positive selection has acted during the evolution of a color gene that seems to be involved in the morphogenesis of a sexually selected trait, the egg-dummies, highlights the importance of further investigations of the comparative genomic basis of the phenotypic diversification of cichlid fishes.

The complete amino acid sequence for brain beta spectrin (beta fodrin): relationship to globin sequences.

The amino acid sequence of mouse brain beta spectrin (beta fodrin), deduced from the nucleotide sequence of complementary DNA clones, reveals that this non-erythroid beta spectrin comprises 2363 residues, with a molecular weight of 274,449 Da. Brain beta spectrin contains three structural domains and we suggest the position of several functional domains including f-actin, synapsin I, ankyrin and spectrin self association sites. Analysis of deduced amino acid sequences indicated striking homology and similar structural characteristics of brain beta spectrin repeats beta 11 and beta 12 to globins. In vitro analysis has demonstrated that heme is capable of specific attachment to brain spectrin, suggesting possible new functions in electron transfer, oxygen binding, nitric oxide binding or heme scavenging.

DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. Importance of the 5'-flanking region.

The three subtypes of the peroxisome proliferator-activated receptors (PPARalpha, beta/delta, and gamma) form heterodimers with the 9-cis-retinoic acid receptor (RXR) and bind to a common consensus response element, which consists of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1). As a first step toward understanding the molecular mechanisms determining PPAR subtype specificity, we evaluated by electrophoretic mobility shift assays the binding properties of the three PPAR subtypes, in association with either RXRalpha or RXRgamma, on 16 natural PPAR response elements (PPREs). The main results are as follows. (i) PPARgamma in combination with either RXRalpha or RXRgamma binds more strongly than PPARalpha or PPARbeta to all natural PPREs tested. (ii) The binding of PPAR to strong elements is reinforced if the heterodimerization partner is RXRgamma. In contrast, weak elements favor RXRalpha as heterodimerization partner. (iii) The ordering of the 16 natural PPREs from strong to weak elements does not depend on the core DR1 sequence, which has a relatively uniform degree of conservation, but correlates with the number of identities of the 5'-flanking nucleotides with respect to a consensus element. This 5'-flanking sequence is essential for PPARalpha binding and thus contributes to subtype specificity. As a demonstration of this, the PPARgamma-specific element ARE6 PPRE is able to bind PPARalpha only if its 5'-flanking region is exchanged with that of the more promiscuous HMG PPRE.

Functional analysis of the 5' flanking sequence of a vaccinia virus late gene.

A series of mutations, including 5' and 3' deletions, as well as insertions were introduced into the 5' flanking nucleotide sequence of a vaccinia virus late gene. This DNA has been shown previously to contain all the necessary elements for correct regulation of the gene most probably transcribed by the viral RNA polymerase. To facilitate the assays, the mutated DNA was fused to the chloramphenicol acetyltransferase gene and inserted into the genome of live vaccinia virus. The effects of the mutations on expression of the chimeric gene were studied by both enzyme assays and nuclease S1 analysis. The results showed that 5' deletions up to about 15 bp from the putative initiation site of transcription still yielded high levels of gene expression. All mutations, however, that deleted the authentic late mRNA start site, abolished promoter activity.

Mitochondrial DNA variation along an altitudinal gradient in the greater white-toothed shrew, Crocidura russula.

The distribution of mitochondrial control region-sequence polymorphism was investigated in 15 populations of Crocidura russula along an altitudinal gradient in western Switzerland. High-altitude populations are smaller, sparser and appear to undergo frequent bottlenecks. Accordingly, they showed a loss of rare haplotypes, but unexpectedly, were less differentiated than lowland populations. Furthermore, the major haplotypes segregated significantly with altitude. The results were inconsistent with a simple model of drift and dispersal. They suggested instead a role for historical patterns of colonization, or, alternatively, present-day selective forces acting on one of the mitochondrial genes involved in metabolic pathways.