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PubMed1997

Host specificity, evolutionary relationships and macrogeographic differentiation among Ascaris populations from humans and pigs.

We describe a variety of restriction site polymorphisms in the introns of Ascaris nuclear genes and in the ribosomal DNA spacers. We use these markers, in addition to previously described mitochondrial variation, to clarify our understanding of the epidemiology of Ascaris in Guatemalan villages where humans and pigs occur in sympatry and to describe the genetic structure of host-associated Ascaris populations from world-wide locations. Intron sequences were amplified from individual worms and alleles defined by endonuclease digestion. Two loci were monomorphic, while 4 length variants and 22 point mutations were detected in the other 7 loci. Within sympatric Guatemalan populations no single locus from either the nuclear or mitochondrial genome was fixed for alternative alleles, although allele frequencies were significantly different at many loci. Phenograms constructed from multilocus nuclear genotypes of individual worms failed to reveal a single case of cross-infection, and demonstrate that divergent mtDNA genotypes are segregating within host-associated populations. On a world-wide scale, the data suggest that extant worm populations result from a single host shift, although characterization of genetic variation in additional loci will be necessary to confirm this. The direction and the geographical origin of the host shift were unresolved. Overall 65% of nuclear genetic variation was found within populations, host (human or pig) explained 18%, while geographical variation within host-associated populations explained 17%. The results (a) demonstrate the utility of introns for studying the epidemiology of parasites showing limited allozyme variation (b) suggest that programmes aiming to control Ascaris infection in the human population can safely ignore zoonotic infection from pigs and (c) illustrate the problems inherent in using single genetic markers to make inferences about the epidemiology of closely related parasite taxa.

Animals↗
PubMed2026

Slow PPi release enhances fidelity of the SARS-CoV-2 RNA dependent RNA polymerase.

Viral RNA-dependent RNA polymerases (RdRps) must balance replication speed with fidelity, preserving genome integrity while permitting enough variability for viral adaptation. The SARS-CoV-2 RdRp complex (non-structural protein 12/7/8) achieves this through the interplay of its intrinsic replication fidelity and a potential proofreading exonuclease complex (NSP10/14). Here, we comprehensively quantify the intrinsic fidelity of the SARS-CoV-2 RdRp through direct pre-steady-state kinetic analyses of nucleotide incorporation across all possible templating bases paired with incoming nucleotides. We discovered substantial variation in discrimination against mismatches ranging from one error in 103 to 108 (median of 105). Crucially, our data reveal a slow pyrophosphate release step that significantly enhances fidelity by effectively introducing a kinetic checkpoint after nucleotide incorporation. The error rates we measured for the RdRp align closely with observed in vivo mutation rates, suggesting that the exonuclease complex may play a less critical role than previously assumed in correcting mistakes during polymerization. These insights advance our understanding of SARS-CoV-2 replication fidelity, and the role of various subcomplexes in genome maintenance and adaptation.

SARS-CoV-2↗
PubMed1994

The suitability of restriction fragment length polymorphism markers for evaluating genetic diversity among and synteny between mosquito species.

Restriction fragment length polymorphism (RFLP) markers derived from the yellow fever mosquito, Aedes aegypti, were used in hybridizations to genomic DNA of the following mosquito species: Ae. albopictus, Ae. togoi, Armigeres subalbatus, Culex pipiens, and Anopheles gambiae. Interspecific hybridization with Ae. aegypti probes varied from 50% (An. gambiae) to 100% (Ae. albopictus) under high stringency conditions. We demonstrated the usefulness of using RFLP profiles to examine genetic diversity between mosquito populations; Ae. aegypti RFLP markers were used to examine genetic relatedness between 10 laboratory strains of Ae. aegypti as well as between nine populations representing four Cx. pipiens subspecies. These results indicate that many Ae. aegypti RFLP markers should have direct applicability in gaining a better understanding of genome structure in other mosquito species, including RFLP linkage mapping and determinations of genetic relatedness among field populations.

Animals↗
PubMed1991

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene.

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.

Amino Acid Sequence↗
PubMed1997

Genetic variation among Mycoplasma agalactiae isolates detected by the variant surface lipoprotein gene (vspA) of Mycoplasma bovis.

Multiple restriction fragments, homologous to the previously described Mycoplasma bovis vspA gene, were identified in the chromosome of Mycoplasma agalactiae. The vspA, a representative variable surface lipoprotein gene of the vsp gene family, and four synthetic oligonucleotides, representing sequences complementary to selected regions of the vsp genes, were used as probes against digested chromosomal DNAs of several M. agalactiae clinical isolates. The resulting Southern blot analysis demonstrated a marked DNA polymorphism of multiple vspA-related fragments among the isolates. An oligonucleotide representing a conserved 5'-region common to all known vsp genes, was found to hybridize to multiple M. agalactiae genomic fragments while the other three oligonucleotides, representing distinct repetitive structures within the coding region of three known vsp genes (vspA, vspE, and vspF), failed to react. These results argue for the possible existence of a gene family in M. agalactiae analogous to the vsp system of M. bovis but comprised of diverse genes.

Antigens, Bacterial↗
PubMed1994

Genes of the linear mitochondrial DNA of Williopsis mrakii: coding sequences for a maturase-like protein, a ribosomal protein VAR1 homologue, cytochrome oxidase subunit 2 and methionyl tRNA.

The mitochondrial DNA (mtDNA) in some yeasts has a linear structure with inverted terminal repeats closed by a single-stranded loop. These mtDNAs have generally a constant gene order, beginning with a small ribosomal RNA gene at the right end and terminating with a cytochrome oxidase subunit 2 gene (COX2) at the left end, independently of the wide variation in genome size. In the mtDNAs from several species of the genus Williopsis, we found an additional open reading frame, ORF1, which was homologous to the Saccharomyces cerevisiae RF1 gene encoding a group I intron maturase-like protein. ORF1 genes from W. mrakii and W. suaveolens were mapped and sequenced. Next to ORF1, COX2 and methionyl tRNA genes were present on the opposite strand. The same relative positions of genes in the mtDNAs so far examined suggests that the constancy of gene order is generally conserved also at the level of individual tRNA genes. We identified another open reading frame, ORF2, in W. mrakii mtDNA. It was mapped next to the cytochrome oxidase subunit 3 gene. Rich in adenine-thymine bases, ORF2 appears to be a homologue of the VAR1 gene which codes for a small ribosomal subunit protein in S. cerevisiae mitochondria. Nucleotide sequences data have been deposited in the EmBL data library under the following Accession Numbers: X66594 (Apocytochrome b and ORF2 genes of W. mrakii), X66595 (ORF1, tRNA-Met and COX2 genes of W. mrakii), X73415 (tRNA-Met and COX2 genes of W. suaveolens), X73416 (ORF1 gene of W. suaveolens) and X73414 (tRNA-Met and COX2 genes of P. jadinii).

Amino Acid Sequence↗
PubMed1990

Cloning and nucleotide sequences of crotamine genes.

A cDNA library containing snake toxin genes was constructed in bacteriophage lambda by using mRNA isolated from the glands of the South American rattlesnake, Crotalus durissus terrificus. The first high-density screening of 400,000 plaques for crotamine-containing genes yielded over 800 positives when a labeled cDNA probe with sequence homology to crotamine was used. Four of these clones with insert sizes from 270 to 400 base pairs were chosen and their inserts subcloned into pGEM-3Z and sequenced. Nucleotide sequence analysis of the cloned cDNAs predicted the existence of multiple variants of the crotamine toxin. The different forms, identified from the DNA sequences, displayed discrepancies in amino acid sequence for crotamine when compared with previously published reports. Direct amino acid sequencing of commercially purified crotamine and CNBr fragments thereof confirmed the structures predicted by the nucleic acid sequences.

Amino Acid Sequence↗
PubMed2006

Archaeal histones and the origin of the histone fold.

Histone sequences have been identified in many archaeal genomes and in environmental samples, and they constitute a family of proteins that are structural homologs of the eukaryotic core histones. Most archaeal histones conform to the single histone-fold structural models that have been described, but a few histone variants exhibit short insertions, additional domains or fusions. Interpretation of these structural variations offers clues to the steps that might have occurred during the evolution and specialization of eukaryotic core histones.

Amino Acid Sequence↗
PubMed2003

Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse.

The nature and organization of polymorphisms, or differences, between genomes of individuals are of great interest, because these variations can be associated with or even underlie phenotypic traits, including disease susceptibility. To gain insight into the genetic and evolutionary factors influencing such biological variation, we have examined the arrangement (haplotype) of single-nucleotide polymorphisms across the genomes of eight inbred strains of mice. These analyses define blocks of high or low diversity, often extending across tens of megabases that are delineated by abrupt transitions. These observations provide a striking contrast to the haplotype structure of the human genome.

Animals↗
PubMed2006

Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method.

We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial 'structural plasticity' and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg2+, Ca2+ and Mn2+ ions. Thus, the functional properties of the variants were dependent on both the structure of their catalytic sites and divalent metal ions performing catalysis.

Base Sequence↗
PubMed2000

Phylogenetic relationships of Pleurotus species according to the sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6 and V9 domains.

A comparative study of the V4, V6 and V9 domains of the mitochondrial small-subunit (SSU) rRNA was conducted to evaluate the use of these sequences to investigate phylogenetic relatedness within the genus Pleurotus. The PCR products encompassing these regions from 48 isolates belonging to 16 Pleurotus species were sequenced and compared. From this comparison, the length and sequence of the three domains were found to be constant within a species. Significant inter-species variations due to insertion/deletion events were found, in most cases occurring in regions not directly involved in the maintainance of the standard SSU rRNA secondary structure. Phylogenetic analysis based upon these mitochondrial sequences was in agreement with relationships previously established by morphological descriptions and with previous studies based upon the nuclear genome or isozymes; moreover such analysis resolved some ambiguities in earlier analyses. It was confirmed that P. ostreatus and P. florida represent a single species, as well as P. pulmonarius and P. sajor-caju. The phylogenetic analysis also made it possible to assess the relative positions of P. rattenburyi, P. lampas, P. sapidus, P. colombinus and P. eryngii. The results clearly showed that sequences of the V4, V6 and V9 domains of the mitochondrial SSU rRNA could provide good markers for use in the taxonomy and phylogeny of species of Basidiomycota. Because of their nucleotide conservation, the major advantage of these species-specific markers was the possibility to study only one isolate from each species to determine phylogenetic relatedness.

Base Sequence↗
PubMed2005

Transposable elements in mosquitoes.

We describe the current state of knowledge about transposable elements (TEs) in different mosquito species. DNA-based elements (class II elements), non-LTR retrotransposons (class I elements), and MITEs (Miniature Inverted Repeat Transposable Elements) are found in the three genera, Anopheles, Aedes and Culex, whereas LTR retrotransposons (class I elements) are found only in Anopheles and Aedes. Mosquitoes were the first insects in which MITEs were reported; they have several LTR retrotransposons belonging to the Pao family, which is distinct from the Gypsy-Ty3 and Copia-Ty1 families. The number of TE copies shows huge variations between classes of TEs within a given species (from 1 to 1000), in sharp contrast to Drosophila, which shows only relatively minor differences in copy number between elements (from 1 to 100). The genomes of these insects therefore display major differences in the amount of TEs and therefore in their structure and global composition. We emphasize the need for more population genetic data about the activity of TEs, their distribution over chromosomes and their frequencies in natural populations of mosquitoes, to further the current attempts to develop a transgenic mosquito unable to transmit malaria that is intended to replace the natural populations.

Animals↗
PubMed2002

From pixels to picograms: a beginners' guide to genome quantification by Feulgen image analysis densitometry.

The study of genome size variation is important from a number of practical and theoretical perspectives. For example, the long-standing "C-value enigma" relating to the more than 200,000-fold range in eukaryotic genome sizes is best studied from a broad comparative standpoint. Genome size data are also required in detailed analyses of genome structure and evolution. The choice of future genome sequencing projects will be dependent on knowledge regarding the sizes of genomes to be sequenced, and so on. To date, genome size data have been acquired primarily by Feulgen microdensitometry or flow cytometry. Each has several advantages but also important limitations. In this review, we provide a practical guide to the new technique of Feulgen image analysis densitometry. The review is designed for those interested in genome size measurements but not extensively experienced in histochemistry, densitometry, or microscopy. Therefore, relevant historical and technical background information is included. For easy reference, we provide recipes for required reagents, guidelines for cell staining, and a checklist of steps for successful image analysis. We hope that the accuracy, rapidity, and cost-effectiveness of Feulgen image analysis demonstrated here will stimulate further surveys of genome sizes in a variety of taxa.

Animals↗
PubMed2000

Genome mapping in ruminants and map locations for genes influencing reproduction.

Genetic maps provide a critical link between genes and phenotypes and are essential tools in the search for the genetic basis of variation in reproductive traits. Genes coding for hormones, growth factors, receptors, binding proteins, transcription factors and enzymes that influence the development and function of the reproductive axis have been assigned to genetic maps of ruminants and locations can be found in the respective genome databases. In addition, comparative information on gene structure and map location will help define the functions of essential genes. Gene locations from other species can be used because of extensive comparative links among mammalian gene maps. Large-scale projects to sequence genes and the ability to map these genes in parallel in radiation hybrid panels of different species will greatly improve the maps and our ability to translate between them. Cloning the genes responsible for genetic differences in fertility and fecundity in ruminants is likely to provide valuable clues to understanding ovarian function and germ cell development.

Animals↗
PubMed1997

Discrimination of hepatitis G virus/GBV-C geographical variants by analysis of the 5' non-coding region.

We have investigated the ability of different subgenomic fragments to reproduce the phylogenetic relationships observed between six complete genome sequences of GBV-C/hepatitis G virus (HGV). While similar relationships were observed following analysis of part of the 5' non-coding region (5'NCR), for the coding region they were not accurately reproduced for some large fragments or for the majority of fragments of 300 or 600 nucleotides. Analysis of 5'NCR sequences from a large number of isolates, including newly obtained sequences from Pakistan, Zaïre and Scotland, produced separate groupings of Asian, African and European/North American variants. These groupings are associated with specific polymorphisms in the 5'NCR, many of which were covariant and consistent with a proposed secondary structure for this region. The relatively low level of amino acid sequence variation observed between these geographically and phylogenetically defined groups of variants suggests that they are unlikely to display significant biological differences.

Base Sequence↗
PubMed1999

Genetic variation maintained in multilocus models of additive quantitative traits under stabilizing selection.

Stabilizing selection for an intermediate optimum is generally considered to deplete genetic variation in quantitative traits. However, conflicting results from various types of models have been obtained. While classical analyses assuming a large number of independent additive loci with individually small effects indicated that no genetic variation is preserved under stabilizing selection, several analyses of two-locus models showed the contrary. We perform a complete analysis of a generalization of Wright's two-locus quadratic-optimum model and investigate numerically the ability of quadratic stabilizing selection to maintain genetic variation in additive quantitative traits controlled by up to five loci. A statistical approach is employed by choosing randomly 4000 parameter sets (allelic effects, recombination rates, and strength of selection) for a given number of loci. For each parameter set we iterate the recursion equations that describe the dynamics of gamete frequencies starting from 20 randomly chosen initial conditions until an equilibrium is reached, record the quantities of interest, and calculate their corresponding mean values. As the number of loci increases from two to five, the fraction of the genome expected to be polymorphic declines surprisingly rapidly, and the loci that are polymorphic increasingly are those with small effects on the trait. As a result, the genetic variance expected to be maintained under stabilizing selection decreases very rapidly with increased number of loci. The equilibrium structure expected under stabilizing selection on an additive trait differs markedly from that expected under selection with no constraints on genotypic fitness values. The expected genetic variance, the expected polymorphic fraction of the genome, as well as other quantities of interest, are only weakly dependent on the selection intensity and the level of recombination.

Alleles↗
PubMed2004

An evaluation of the current state of genomic data privacy protection technology and a roadmap for the future.

The incorporation of genomic data into personal medical records poses many challenges to patient privacy. In response, various systems for preserving patient privacy in shared genomic data have been developed and deployed. Although these systems de-identify the data by removing explicit identifiers (e.g., name, address, or Social Security number) and incorporate sound security design principles, they suffer from a lack of formal modeling of inferences learnable from shared data. This report evaluates the extent to which current protection systems are capable of withstanding a range of re-identification methods, including genotype-phenotype inferences, location-visit patterns, family structures, and dictionary attacks. For a comparative re-identification analysis, the systems are mapped to a common formalism. Although there is variation in susceptibility, each system is deficient in its protection capacity. The author discovers patterns of protection failure and discusses several of the reasons why these systems are susceptible. The analyses and discussion within provide guideposts for the development of next-generation protection methods amenable to formal proofs.

Computer Security↗
PubMed1999

Effect of cytarabine on the NMR structure of a model okazaki fragment from the SV40 genome.

Okazaki fragments occur as intermediates during lagging strand DNA replication. Alterations in Okazaki fragment structure may contribute to the anticancer activities of nucleoside analogues such as cytarabine, a potent anti-leukemic agent that inhibits lagging strand replication. We have determined the solution structures for two model Okazaki fragments, [OKA] and [ARAC]. These sequences are derived from a frequent initiation site for primase during replication of the SV-40 viral genome. The sequence of [ARAC] differs from [OKA] only by substitution of cytarabine for one deoxycytidine. The structure of each model Okazaki fragment was elucidated using NMR spectroscopy and restrained molecular dynamics simulations. The solution structures of [OKA] and [ARAC] each consist of two distinct domains: a DNA duplex region (DDR) and an RNA-DNA hybrid duplex region (HDR). The DDR of [OKA] adopts geometry similar to B-form except for variations in helical parameters, especially twist and roll, which occur in the purine tract, increasing base overlap among the five consecutive purines. The helical axes for the DDR and HDR of [OKA] are bent 22 degrees relative to one another. Although the local structures for the DDR and HDR of [ARAC] are similar to those in [OKA] (root-mean-square deviation (rmsd) approximately 0.8, 1.7 A), the bending at the junction is different (41 degrees for [ARAC] vs 22 degrees for [OKA]). Increased helical bending of cytarabine-substituted Okazaki fragments may contribute to the propensity of cytarabine to inhibit elongation of the lagging strand during DNA replication, and in effecting anticancer activity.

Algorithms↗