Guy Drouin

Relative rates of synonymous substitutions in the mitochondrial, chloroplast and nuclear genomes of seed plants

Previous studies have estimated that, in angiosperms, the synonymous substitution rate of chloroplast genes is three times higher than that of mitochondrial genes and that of nuclear genes is twelve times higher than that of mitochondrial genes. Here we used 12 genes in 27 seed plant species to investigate whether these relative rates of substitutions are common to diverse seed plant groups. We find that the overall relative rate of synonymous substitutions of mitochondrial, chloroplast and nuclear genes of all seed plants is 1:3:10, that these ratios are 1:2:4 in gymnosperms but 1:3:16 in angiosperms and that they go up to 1:3:20 in basal angiosperms. Our results show that the mitochondrial, chloroplast and nuclear genomes of seed plant groups have different synonymous substitutions rates, that these rates are different in different seed plant groups and that gymnosperms have smaller ratios than angiosperms.

Diffusion coefficient of DNA molecules during free solution electrophoresis.

The free‐draining properties of DNA normally make it impossible to separate nucleic acids by free‐flow electrophoresis. However, little is known, either theoretically or experimentally, about the diffusion coefficient of DNA molecules during free‐flow electrophoresis. In fact, many authors simply assume that the Nernst‐Einstein relation between the mobility and the diffusion coefficient still holds under such conditions. In this paper, we present an experimental study of the diffusion coefficient of both ssDNA and dsDNA molecules during free‐flow electrophoresis. Our results unequivocally show that a simplistic use of Nernst‐Einsteins relation fails, and that the electric field actually has no effect on the thermal diffusion process. Finally, we compare the dependence of the diffusion coefficient upon DNA molecular size to results obtained previously by other groups and to Zimms theory.

The Giardia lamblia actin gene and the phylogeny of eukaryotes.

The single-copy actin gene of Giardia lamblia lacks introns; it has an average of 58% amino acid identity with the actin of other species; and 49 of its amino acids can be aligned with the amino acids of a consensus sequence of heat shock protein 70. Analysis of the potential RNA secondary structure in the transcribed region of the G. lamblia actin gene and of the single-copy actin gene of nine other species did not reveal any conserved structures. The G. lamblia actin sequence was used to root the phylogenetic trees based on 65 actin protein sequences from 43 species. This tree is congruent with small-subunit rRNA trees in that it shows that oomycetes are not related to higher fungi; that kinetoplatid protozoans, green plants, fungi and animals are monophyletic groups; and that the animal and fungal lineages share a more recent common ancestor than either does with the plant lineage. In contrast to smalls-ubunit rRNA trees, this tree shows that slime molds diverged after the plant lineage. The slower rate of evolution of actin genes of slime molds relative to those of plants, fungi, and animals species might be responsible for this incongruent branching.

Characterization of the gene conversions between the multigene family members of the yeast genome

Stanley Sawyers gene conversion detection method, implemented in his GENECONV computer program, was used to detect and characterize the gene conversions between the multigene family members of the yeast genome. This method gave different gene conversion frequencies and size distribution for gene families with two members and multigene families with more than two members. The 69 gene conversions detected in multigene families with more than two members occur at a frequency of 7.8% gene conversion/pair of genes compared and have an average size of 173+/-220 nucleotides. Larger gene conversions are found only between more similar genes, the genes involved in gene conversions are distributed almost randomly among the 16 yeast chromosomes, and the frequency of gene conversions increases as the distance between repeated genes decreases. In contrast to previous studies, no relationship was observed between the level of expression of a gene and its involvement in gene conversions. These analyses also suggest that gene conversions might occur by different mechanisms in closely linked genes and unlinked genes. The excess of converted regions at the 3? end of unlinked genes suggests that recombination with incomplete cDNA molecules is the main mechanism responsible for gene conversions between such genes.

Free-solution electrophoresis of DNA

We report the first experimental evidence that double-stranded DNA fragments can be electrophoretically separated in free-solution (i.e., in the absence of a sieving matrix) when either a single or two streptavidin molecules are attached to the end(s) using biotinylated nucleotides. As previously predicted, higher resolution is obtained at higher electric fields or when two streptavidin molecules are attached to each DNA fragment. The resolution is also affected by the diameter and coating of the capillaries.

Separating DNA sequencing fragments without a sieving matrix

The possibility of separating appropriately labeled DNA fragments using free‐flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free‐flow separation of double‒stranded DNA (dsDNA) fragments in the 100—1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end‒labeled free‒flow electrophoresis (ELFSE) can also be used to sequence single‒stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5′‒end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte‒wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.

Inactivation dates of the human and guinea pig vitamin C genes

The capacity to biosynthesize ascorbic acid has been lost in a number of species including primates, guinea pigs, teleost fishes, bats, and birds. This inability results from mutations in the GLO gene coding for L-gulono-γ-lactone oxidase, the enzyme responsible for catalyzing the last step in the vitamin C biosynthetic pathway. We analyzed available primate and rodent GLO gene sequences to determine their evolutionary history. We used a method based on sequence comparisons of lineages with and without functional GLO genes to calculate inactivation dates of 61 and 14 MYA for the primate and guinea pig genes, respectively. These estimates are consistent with previous phylogeny-based estimates. An analysis of transposable element distribution in the primate and rodent GLO sequences did not reveal conclusive evidence that illegitimate recombination between repeats has contributed to the loss of exons in the primate and guinea pig genes.

Processed Pseudogenes, Processed Genes, and Spontaneous Mutations in the Arabidopsis Genome

We identified 411 processed sequences in the Arabidopsis thaliana genome based on the fact that they have lost their intron(s) and have a length that is at least 95% of the length of the gene that gave rise to them. These sequences were generated by 230 different genes and clearly originated from retrotranspositons events because most of them (91%) have a poly(A)-tail. They are composed of 376 sequences with frame shifts and/or premature stop codons (processed pseudogenes) and 35 sequences without disablements (processed genes). Eleven of these processed genes are likely functional retrotransposed genes because they have low Ka/Ks ratios and high Ks values, and their sequences match numerous Arabidopsis ESTs. Processed sequences are mostly randomly distributed in the Arabidopsis genome and their rate of accumulation has steadily been decreasing since it peaked some 50 MYA. In contrast with the situation observed in mammals, the processed sequences found in the Arabidopsis genome originate from genes with high copy numbers and not from highly expressed genes. The patterns of spontaneous mutations in Arabidopsis are slightly different than those of mammals but are similar to those observed in Drosophila. This suggests that methylated cytosine deamination is less frequent in Arabidopsis than in mammals.

Gel electrophoretic mobility of single-stranded DNA: the two reptation field-dependent factors.

The reptation model is the dominant theory in understanding the electrophoretic separation of single‐stranded DNA molecules in gels or entangled polymer solutions. Recently, we showed that the Ogston and reptation regimes are separated by an entropic trapping regime at low field intensities. Here, we report the first comparison of the field‐dependent part of the DNA mobility for both small and long reptating molecules. We show that both mobilities increase linearly with field intensity, with the mobility of the longer (comigrating) fragments increasing faster than that of the smaller ones. We compare our results to the predictions of the biased reptation model.

MIGRATION OF DNA THROUGH GELS

Publisher Summary Gel electrophoresis has become a major laboratory tool for separating biological macromolecules. For example, large (megabase) double-stranded DNA (dsDNA) molecules can readily be separated on agarose gels, using pulsed-field gel electrophoresis (PFGE), while subkilobase single-stranded DNA (ssDNA) molecules can be sequenced on polyacrylamide gels. These two techniques are essential to map and sequence the human genome. This chapter first discusses the different models of DNA migration through gels, and then their limitations and the conditions, under which they should apply. It then describes how experimental data should be obtained and analyzed in order to (1) identify the migration mechanism that is taking place, (2) estimate the parameters of the relevant model, and (3) optimize separation. As an example, it applies this approach to the separation (sequencing) of ssDNA in polyacrylamide gels. It concludes, by briefly reviewing the challenges still open in the study of DNA sequencing, and briefly introduces some new ideas being explored.