Determinants of rate variation in mammalian DNA sequence evolution

Attempts to analyze variation in the rates of molecular evolution among mammalian lineages have been hampered by paucity of data and by nonindependent comparisons. Using phylogenetically independent comparisons, we test three explanations for rate variation which predict correlations between rate variation and generation time, metabolic rate, and body size. Mitochondrial and nuclear genes, protein coding, rRNA, and nontranslated sequences from 61 mammal species representing 14 orders are used to compare the relative rates of sequence evolution. Correlation analyses performed on differences in genetic distance since common origin of each pair against differences in body mass, generation time, and metabolic rate reveal that substitution rate at fourfold degenerate sites in two out of three protein sequences is negatively correlated with generation time. In addition, there is a relationship between the rate of molecular evolution and body size for two nuclear-encoded sequences. No evidence is found for an effect of metabolic rate on rate of sequence evolution. Possible causes of variation in substitution rate between species are discussed.     

Structural genes adjacent to interspersed repetitive DNA sequences

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. ³H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3–5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80% of that mRNA hybridizable with whole DNA (approximately 65%) had reacted with the repeat-contiguous DNA fraction (50%). This result shows that 80–100% of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.     

Understanding human DNA sequence variation

Over the past century researchers have identified normal genetic variation and studied that variation in diverse human populations to determine the amounts and distributions of that variation. That information is being used to develop an understanding of the demographic histories of the different populations and the species as a whole, among other studies. With the advent of DNA-based markers in the last quarter century, these studies have accelerated. One of the challenges for the next century is to understand that variation. One component of that understanding will be population genetics. We present here examples of many of the ways these new data can be analyzed from a population perspective using results from our laboratory on multiple individual DNA-based polymorphisms, many clustered in haplotypes, studied in multiple populations representing all major geographic regions of the world. These data support an "out of Africa" hypothesis for human dispersal around the world and begin to refine the understanding of population structures and genetic relationships. We are also developing baseline information against which we can compare findings at different loci to aid in the identification of loci subject, now and in the past, to selection (directional or balancing). We do not yet have a comprehensive understanding of the extensive variation in the human genome, but some of that understanding is coming from population genetics.     

Nuclear ribosomal DNA sequence variation and evolution of spotted marsh-orchids (Dactylorhiza maculata group)

Sequences of both internal and external transcribed spacers of nuclear ribosomal DNA were sequenced for four species belonging to the Dactylorhiza maculata group or "spotted marsh-Orchids". These four species are D. fuchsii, D. saccifera, D. foliosa, and D. maculata. Extensive nuclear ribosomal DNA polymorphism was uncovered within the diploid D. fuchsii and the putative autotetraploid D. maculata. Within the phylogenetic trees reconstructed using parsimony and Bayesian analyses, four main lineages (A, B, C, and D) were well supported. While D. saccifera, D. maculata, and D. foliosa were confined to clades B, C, and D, respectively, D. fuchsii accessions were spread over three clades (A, B, and C). Lineage C, which included accessions of the diploid D. fuchsii and the tetraploid D. maculata, was closely related to the lineage of D. foliosa (lineage D), an endemic diploid species from Madeira. Moreover, intra-individual polymorphism was found within accessions of D. maculata, D. fuchsii, and D. saccifera. It is shown that in some instances two lineages, contributed to the observed intra-individual polymorphism (C and A in D. maculata, A and B in D. fuchsii and D. saccifera). Evolutionary scenarios leading to this extensive nuclear ribosomal DNA polymorphism are discussed in the light of results from maternally inherited chloroplast DNA markers and an autopolyploid origin of D. maculata from a D. foliosa-like ancestor is postulated.     

Functional variation and evolution of non-coding DNA

The focus of large genomic studies has shifted from only looking at genes and protein-coding sequences to exploring the full set of elements in each genome. The explosion of comparative sequencing data has led to an increase in methodologies, approaches and ideas on how to analyze the unknown fraction of the genome, namely the non-protein-coding fraction. The main issues relate to the discovery, evolutionary analysis and natural variation of non-coding DNA, and the parameters that prevent us from fully understanding the properties of non-coding DNA.     

Human-chimpanzee DNA sequence variation in the four major genes of the renin angiotensin system

The renin angiotensin system (RAS) is involved in blood pressure control and water/sodium metabolism. The genes encoding the proteins of this system are candidate genes for essential hypertension. The RAS involves four main molecules: angiotensinogen, renin, angiotensin I-converting enzyme, and the angiotensin II type 1 receptor (encoded by the genes AGT, REN, DCP1, and AGTR1, respectively). We performed a molecular screening over 17,037 bp of the coding and 5' and 3' untranslated regions of these genes, from three to six common chimpanzees. We identified 44 single-nucleotide polymorphisms (SNPs) in chimpanzee samples, including 18 coding-region SNPs, 5 of which led to an amino acid replacement. We observed common and different features at various sites (synonymous, nonsynonymous, and noncoding) within and between the four chimpanzee genes: (1) the nucleotide diversity at noncoding sites was similar; (2) the nucleotide diversity at nonsynonymous sites was low, probably reflecting purifying selection, except for the AGT gene; (3) the nucleotide diversity at synonymous sites, which was dependent on the G+C content at the third position of the codon, was high, except for the AGTR1 gene. Comparison of the chimpanzee SNPs with those previously reported for humans identified 119 sites with fixed differences (including 62 coding sites, 17 of which resulted in amino acid differences between the species). Analysis of polymorphism within species and divergence between species shed light on the evolutionary constraints on these genes. In particular, comparison of the pattern of mutation at polymorphic and fixed sites between humans and chimpanzees suggested that the high G+C content of the DCP1 gene was maintained by positive selection at its silent sites. Finally, we propose 68 ancestral alleles for the human RAS genes and discuss the implications for their use in future hypertension-susceptibility association studies.     

A model of DNA sequence evolution

Statistical studies of gene populations on the purine/pyrimidine alphabet have shown that the mean occurrence probability of thei-motif YRY(N) _i YRY (R=purine, Y=pyrimidine, N=R or Y) is not uniform by varyingi in the range [1,99], but presents a maximum ati=6 in the following populations: protein coding genes of eukaryotes, prokaryotes, chloroplasts and mitrochondria, and also viral introns, ribosomal RNA genes and transfer RNA genes (Arquès and Michel, 1987b,J. theor. Biol. 128, 457–461). From the “universality” of this observation, we suggested that the oligonucleotide YRY(N)₆ is a primitive one and that it has a central function in DNA sequence evolution (Arquès and Michel, 1987b,J. theor. Biol. 128, 457–461). Following this idea, we introduce a concept of a model of DNA sequence evolution which will be validated according to a shema presented in three parts. In the first part, using the last version of the gene database, the YRY(N)₆YRY preferential occurrence (maximum ati=6) is confirmed for the populations mentioned above and is extended to some newly analysed populations: chloroplast introns, chloroplast 5′ regions, mitochondrial 5′ regions and small nuclear RNA genes. On the other hand, the YRY(N)₆YRY preferential occurrence and periodicities are used in order to classify 18 gene populations. In the second part, we will demonstrate that several statistical features characterizing different gene populations (in particular the YRY(N)₆YRY preferential occurrence and the periodicities) can be retrieved from a simple Markov model based on the mixing of the two oligonucleotides YRY(N)₆ and YRY(N)₃ and based on the percentages of RYR and YRY in the unspecified trinucleotides (N)₃ of YRY(N)₆ and YRY(N)₃. Several properties are identified and prove in particular that the oligonucleotide mixing is an independent process and that several different features are functions of a unique parameter. In the third part, the return of the model to the reality shows a strong correlation between reality and simulation concerning the presence of large alternating purine/pyrimidine stretches and of periodicities. It also contributes to a greater understanding of biological reality, e.g. the presence or the absence of large alternating purine/pyrimidine stretches can be explained as being a simple consequence of the mixing of two particular oligonucleotides. Finally, we believe that such an approach is the first step toward a unified model of DNA sequence evolution allowing the molecular understanding of both the origin of life and the actual biological reality.     

MVR-PCR analysis of hypervariable DNA sequence variation

Techniques for accurate marking of infectious microbial agents circulating in populations would be very useful to epidemiologists. In this article, David Arnot, Cally Roper and Ali Sultan review recent progress in transferring MVR-PCR DNA finger-printing techniques from human forensic medicine to parasitology.     

Allelic variation adjacent to the human insulin and apolipoprotein C-II genes in different ethnic groups

Summary We have used DNA probes for the human insulin gene and apolipoprotein C-II (apo C-II) gene to determine the extent of allelic variation in different ethnic groups. The distribution of an apo C-II DNA polymorphism revealed by the restriction endonuclease Taq I showed no significant variation amongst racial groups; in contrast, an insulin gene-related DNA polymorphism showed marked variability. In Japanese, Chinese, and Asian Indian groups there was an increased frequency of homozygosity for the class 1 allele compared to Caucasian groups (P<0.001, P<0.01, and P<0.05, respectively). In Caucasian, Japanese, Chinese, and Asian Indian groups no class 2 allele was observed; but in the Negroid populations (African and West Indian) the class 2 allele frequencies were 0.23 and 0.25 respectively. Possible reasons for this variation in allele distribution are considered in relation to disease associations.     

Survey of polymorphic sequence variation in the immediate 5' region of human DNA repair genes

Systematic screens have revealed extensive DNA sequence variation existing in the human population. Studies of the role of polymorphic genetic variants in explaining the association of family history with risk of common disease have generally focused on variants predicted to disrupt protein structure and activity. Recent studies have identified genetic variation in the level of expression of many genes, variation that is potentially biologically relevant in explaining individual variation in disease risk. In a survey of data available for 108 DNA repair genes that have been systematically screened for sequence variation, an average of 3.3 SNPs per gene were found to exist at a variant allele frequency of at least 0.02 in the region 2kb upstream from the 5'-untranslated region. One-third of the genes harbored a SNP with an allele frequency of at least 0.02 within a predicted promotor element. These variants are distributed among promoter elements that average 20 elements per gene. The frequency of polymorphic SNPs in CpG islands was 0.8 per gene, while the frequency of SNPs in the 5'-UTR was 0.7 per gene. The recognition of extensive genetic variation with potential to impact levels of gene expression, and thereby exacerbate the impact of amino acid substitution variants on the activity of proteins, increases the complexity of analyses required to explain the molecular genetic basis for the familial contribution to the sporadic incidence of common disease.     

Mitochondrial DNA variation in human evolution and disease

Analysis of mitochondrial DNA (mtDNA) variation has permitted the reconstruction of the ancient migrations of women. This has provided evidence that our species arose in Africa about 150000 years before present (YBP), migrated out of Africa into Asia about 60000 to 70000 YBP and into Europe about 40000 to 50000 YBP, and migrated from Asia and possibly Europe to the Americas about 20000 to 30000 YBP. Although much of the mtDNA variation that exists in modern populations may be selectively neutral, studies of the mildly deleterious mtDNA mutations causing Leber's hereditary optic neuropathy (LHON) have demonstrated that some continent-specific mtDNA lineages are more prone to manifest the clinical symptoms of LHON than others. Hence, all mtDNA lineages are not equal, which may provide insights into the extreme environments that were encountered by our ancient ancestor, and which may be of great importance in understanding the pathophysiology of mitochondrial disease.     

High-accuracy DNA sequence variation screening by DHPLC

Genetic maps based on biallelic single-nucleotide polymorphisms amenable to microarray-based genotyping have significantly accelerated the mapping of mono- and multigenic traits in model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana. This advance needs to be matched by highly accurate, inexpensive and robust methodology for fine-structure mapping of the candidate region(s) and the eventual identification of the causative mutation(s). To establish the usefulness of denaturing high-performance liquid chromatography (DHPLC) for those purposes, we have amplified 476 fragments from two A. thaliana ecotypes with an average length of 563 bp covering various candidate regions on chromosomes 1, 2 and 4. Parallel analysis by DHPLC and dye terminator sequencing showed that DHPLC detected 165 out of 166 polymorphic fragments with only four false positives, amounting to a sensitivity, specificity and accuracy of 99.4%, 98.7% and 99%, respectively. It proved beneficial to analyze the fragments not only at the highest but also at the lower temperatures recommended by the algorithm freely available at http:?insertion.stanford.edu/melt.html.     

Mitochondrial DNA sequence variation in Greeks.

Mitochondrial DNA (mtDNA) control region sequences were determined in 54 unrelated Greeks, coming from different regions in Greece, for both segments HVR-I and HVR-II. Fifty-two different mtDNA haplotypes were revealed, one of which was shared by three individuals. A very low heterogeneity was found among Greek regions. No one cluster of lineages was specific to individuals coming from a certain region. The average pairwise difference distribution showed a value of 7.599. The data were compared with that for other European or neighbor populations (British, French, Germans, Tuscans, Bulgarians, and Turks). The genetic trees that were constructed revealed homogeneity between Europeans. Median networks revealed that most of the Greek mtDNA haplotypes are clustered to the five known haplogroups and that a number of haplotypes are shared among Greeks and other European and Near Eastern populations.     

DNA sequence and haplotype variation in two candidate genes for dilated cardiomyopathy in the turkey Meleagris gallopavo

Determining variation in genes is fundamental to understanding their function in the disease state. Cardiac troponin T (cTnT) and phospholamban (PLN) genes have been implicated in dilated cardiomyopathy (DCM) in human and model species. To investigate the role of these 2 candidate genes in DCM in the turkey Meleagris gallopavo, understanding sequence variants and map position distribution is necessary. To this end, a total of 1854 and 1771 bp of cTnT and PLN gene sequences, respectively, were scanned for single nucleotide polymorphisms (SNPs) in a randomly bred population. A total of 15 SNPs was identified in the cTnT and PLN genomic sequences. Nine haplotypes, 5 in cTnT and 4 in PLN, were identified. Observed heterozygosities (0.02-0.39) in the turkey population were low for both genes. Within each gene, 1 SNP corresponding to a restriction enzyme site was identified and used to develop a PCR-restriction fragment length polymorphism (RFLP) genotyping assay. The PLN gene was genetically mapped to turkey chromosome 2, equivalent to Gallus gallus chromosome 3, and cTnT mapped to a turkey microchromosome. Although limited because of the relatively small sample size of 55 birds, the data from this SNP analysis of PLN and cTnT provide a foundation from which to evaluate the function of cTnT and PLN in the turkey. Information about the distribution of the SNPs and haplotypes will facilitate future association and linkage studies.     

Homology between the invertible deoxyribonucleic acid sequence that controls flagellar-phase variation in Salmonella sp. and deoxyribonucleic acid sequences in other organisms.

The invertible deoxyribonucleic acid (DNA) segment cloned from Salmonella sp. was radioactively labeled and used as a probe to search for homologous sequences by Southern hybridization. Only one copy of the invertible segment could be found on the Salmonella sp. genome. Partial sequence homology with the invertible region was detected in bacteriophage Mu and P1 DNA by low-stringency hybridization. Under these conditions, no homology was detected with Escherichia coli DNA. A strain of Salmonella sp. defective in phase variation carrying the vH2- allele was also analyzed by DNA-DNA hybridization. The results show that there is sequence divergence between diphasic and vH2- strains within the invertible sequence.     

Carp preproinsulin cDNA sequence and evolution of insulin genes.

The nucleic acid sequence of the preproinsulin cDNA of carp (Cyprinus carpio), cloned in the PstI-site of pBR322 (1), has been determined. The sequenced insert of 439 bp includes the complete coding information for carp preproinsulin (108 amino acids), 10 nucleotides of the 5'-and 105 nucleotides of the 3'-nontranslated regions. The nucleotide sequence confirms the previously established amino acid sequence of carp insulin (2) and determines those of the signal (21 aa 1) and C-peptide (35 aa 1). The observed shortness of the signal peptide of carp preproinsulin and the N-terminal addition of 2 amino acids to the carp insulin B-chain suggest that the cleavage site of the signal peptidase has moved. Calculations based on the comparison of known preproinsulin cDNA sequences showed that the evolutionary distance between fresh water and salt water teleostians is not smaller than between man and chicken.     

Two DNA invertases contribute to flagellar phase variation in Salmonella enterica serovar Typhimurium strain LT2

Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene fin. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. The hin disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the fin disruption mutant could undergo phase variation, fin hin double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring hin mutation, lacks Fels-2, which ensures the phase stability in this serovar.