Evolution of Microsatellite Alleles in Four Species of Mice (Genus Apodemus)

Microsatellite length variation was investigated at a highly variable microsatellite locus in four species of Apodemus. Information obtained from microsatellite allele sequences was contrasted with allele sizes, which included 18 electromorphs. Additional analysis of a 400-bp unique sequence in the flanking region identified 26 different haplotype sequences or ``true' alleles in the sample. Three molecular mechanisms, namely, (1) addition/deletion of repeats, (2) substitutions and indels in the flanking region, and (3) mutations interrupting the repeat, contributed to the generation of allelic variation. Size homoplasy can be inferred for alleles within populations, from different populations of the same species, and from different species. We propose that microsatellite flanking sequences may be informative markers for investigating mutation processes in microsatellite repeats as well as phylogenetic relationships among alleles, populations, and species. Received: 3 November 1999 / Accepted: 2 May 2000     

Differential selective pressures on the merozoite surface protein 2 locus of Plasmodium falciparum in a low endemic area

195 Plasmodium falciparum merozoite surface protein-2 alleles collected in Tak Province, Thailand, in 1996 and 2006 revealed extremely limited sequence polymorphism except in the variable (V) region, which defines the two allelic families 3D7 and FC27. This pattern is most easily explained by repeated inter-allelic gene conversion events homogenizing alleles outside the V region. Comparison of synonymous and nonsynonymous differences in V regions within allelic families supported the hypothesis that amino acid sequence polymorphism in this region is selectively favored. The pattern of sequence differentiation supported the hypothesis that repeats in the V region have evolved by concerted evolution in the 3D7 family but not in the FC27 family. In the FC27 family two alleles of relatively high frequency were the most common V-region alleles in both 1996 and 2006, while 3D7 alleles constituted a significantly greater proportion of the sequences collected in 2006 (56.1%) than of those collected in 1996 (28.9%). These changes in the frequencies of 3D7 alleles may reflect increased intensity of selection on the P. falciparum population in Thailand as a result of effective control measures that have sharply reduced the incidence of malaria infection.     

Phylogenetic Analysis of the Friedreich Ataxia GAA Trinucleotide Repeat

Friedreich ataxia is an autosomal recessive neurodegenerative disorder associated with a GAA repeat expansion in the first intron of the gene (FRDA) encoding a novel, highly conserved, 210 amino acid protein known as frataxin. Normal variation in repeat size was determined by analysis of more than 600 DNA samples from seven human populations. This analysis showed that the most frequent allele had nine GAA repeats, and no alleles with fewer than five GAA repeats were found. The European and Syrian populations had the highest percentage of alleles with 10 or more GAA repeats, while the Papua New Guinea population did not have any alleles carrying more than 10 GAA repeats. The distributions of repeat sizes in the European, Syrian, and African American populations were significantly different from those in the Asian and Papua New Guinea populations (p < 0.001). The GAA repeat size was also determined in five nonhuman primates. Samples from 10 chimpanzees, 3 orangutans, 1 gorilla, 1 rhesus macaque, 1 mangabey, and 1 tamarin were analyzed. Among those primates belonging to the Pongidae family, the chimpanzees were found to carry three or four GAA repeats, the orangutans had four or five GAA repeats, and the gorilla carried three GAA repeats. In primates belonging to the Cercopithecidae family, three GAA repeats were found in the mangabey and two in the rhesus macaque. However, an AluY subfamily member inserted in the poly(A) tract preceding the GAA repeat region in the rhesus macaque, making the amplified sequence approximately 300 bp longer. The GAA repeat was also found in the tamarin, suggesting that it arose at least 40 million years ago and remained relatively small throughout the majority of primate evolution, with a punctuated expansion in the human genome. Received: 18 August 2000 / Accepted: 10 November 2000     

Microsatellite Allelic Homoplasy Due to Variable Flanking Sequences

Microsatellite DNA sequences have become the dominant source of nuclear genetic markers for most applications. It is important to investigate the basis of variation between alleles and to know if current assumptions about the mechanisms of microsatellite mutation (that is to say, variations involving simple changes in the number of repeat) are correct. We have characterized, by DNA sequencing, the human alleles of a new highly informative (CA)n repeat localized approximately 20 kb centromeric to the HLA-B gene. Although 12 alleles were identified based on conventional length criteria, sequencing of the alleles demonstrated that differences between alleles were found to be more complex than previously assumed: A high degree of microsatellite variability is due to variation in the region immediately flanking the repeat. These data indicate that the mutational process which generates polymorphism in this region has involved not only simple changes in the number of dinucleotide CA repeats but also perturbations in the nonrepeated 5′ and 3′ flanking sequences. Three families of alleles (not visible from the overall length of the alleles), with presumably separate evolutionary histories, exist and can yield to homoplasy of size. Effectively, we can observe alleles of the same size with different internal structures which are separated by a significant amount of variation. Although allelic homoplasy for noninterrupted microsatellite loci has been suggested between different species, it has not been unequivocally demonstrated within species. A strong association is noted between alleles defined at the sequence level and HLA-B alleles. The observation of several families of alleles at the population level provides information about the evolutionary history and mutation processes of microsatellites and may have implications for the use of these markers in phylogenetic, linkage disequilibrium studies, and gene mapping. Received: 14 May 1996 / Accepted: 9 September 1996     

Evolution of Tandemly Repeated Sequences: What Happens at the End of an Array?

Tandemly repeated sequences are a major component of the eukaryotic genome. Although the general characteristics of tandem repeats have been well documented, the processes involved in their origin and maintenance remain unknown. In this study, a region on the paternal sex ratio (PSR) chromosome was analyzed to investigate the mechanisms of tandem repeat evolution. The region contains a junction between a tandem array of PSR2 repeats and a copy of the retrotransposon NATE, with other dispersed repeats (putative mobile elements) on the other side of the element. Little similarity was detected between the sequence of PSR2 and the region of NATE flanking the array, indicating that the PSR2 repeat did not originate from the underlying NATE sequence. However, a short region of sequence similarity (11/15 bp) and an inverted region of sequence identity (8 bp) are present on either side of the junction. These short sequences may have facilitated nonhomologous recombination between NATE and PSR2, resulting in the formation of the junction. Adjacent to the junction, the three most terminal repeats in the PSR2 array exhibited a higher sequence divergence relative to internal repeats, which is consistent with a theoretical prediction of the unequal exchange model for tandem repeat evolution. Other NATE insertion sites were characterized which show proximity to both tandem repeats and complex DNAs containing additional dispersed repeats. An ``accretion model' is proposed to account for this association by the accumulation of mobile elements at the ends of tandem arrays and into ``islands' within arrays. Mobile elements inserting into arrays will tend to migrate into islands and to array ends, due to the turnover in the number of intervening repeats. Received: 18 August 1997 / Accepted: 18 September 1998     

Mutational mechanisms,phylogeny, and evolution of a repetitive region within a clock gene ofDrosophila melanogaster

The D. melanogaster clock gene period (per) is an internally repetitive gene encoding a tandem array of Thr-Gly codons that are highly polymorphic in length in European natural populations. The two major length variants, (Thr-Gly)₂₀ and (Thr-Gly)₁₇, show a highly significant latitudinal cline. In this study we present the complete sequence of the Thr-Gly region of 91 individuals from 6 natural populations of D. melanogaster, 5 from Europe and 1 from North Africa. We further characterized these 91 individuals for polymorphic sites in two other regions, one upstream and one downstream of the Thr-Gly repeat. We used the haplotypic combinations of Thr-Gly allele with flanking markers in an attempt to identify the mechanisms involved in the evolution of the D. melanogaster Thr-Gly region and to infer the phylogenetic relationship existing among the Thr-Gly alleles. We observe evidence for both intra- and interallelic mutational mechanisms, including replication slippage, unequal crossing-over, and gene conversion. Received: 22 August 1995 / Accepted: 17 October 1995     

Mhc Allelic Diversity and Modern Human Origins

Thirty complete coding sequences of human major histocompatibility complex (Mhc) class II DRB alleles, spanning 237 codons, were analyzed for phylogenetic information using distance, parsimony, and likelihood approaches. Allelic genealogies derived from different parts of the coding sequence (exon 2, the 5′ and 3′ ends of exon 2, respectively, and exons 3–6) were compared. Contrary to prior assertions, a rigorous analysis of allelic genealogies in this gene family cannot be used to justify the claim that the lineage leading to modern humans contained on average at least 100,000 individuals. Phylogenetic inferences based upon the exon 2 region of the DRB loci are complicated by selection and recombination, so this part of the gene does not provide a complete and accurate view of allelic relationships. Attempts to reconstruct human history from genetic data must use realistic models which consider the complicating factors of nonequilibrium populations, recombination, and different patterns of selection. Received: 19 February 1997 / Accepted: 12 June 1997     

Extensive Amplification and Transposition of a Novel Repetitive Element, Xstir, Together with Its Terminal Inverted Repeat in the Evolution of Xenopus

A DNA fragment containing short tandem repeat sequences (approximately 86-bp repeat) was isolated from a Xenopus laevis cDNA library. Southern blot and in situ hybridization analyses revealed that the repeat was highly dispersed in the genome and was present at approximately 1 million copies per haploid genome. We named this element Xstir (Xenopus short tandemly and invertedly repeating element) after its arrangement in the genome. The majority of the genomic Xstir sequences were digested to monomer and dimer sizes with several restriction enzymes. Their sequences were found to be highly homogeneous and organized into tandem arrays in the genome. Alignment analyses of several known sequences showed that some of the Xstir-like sequences were also organized into interspersed inverted repeats. The inverted repeats consisted of an inverted pair of two differently modified Xstirs separated by a short insert. In addition, these were framed by another novel inverted repeat (Xstir-TIR). The Xstir-TIR sequence was also found at the ends of tandem Xstir arrays. Furthermore, we found that Xstir-TIR was linked to a motif characterizing the T2 family which belonged to a vertebrate MITE (miniature inverted-repeat transposable element) family, suggesting the importance of Xstir-TIR for their amplification and transposition. The present study of 11 anuran and 2 urodele species revealed that Xstir or Xstir-like sequences were extensively amplified in the three Xenopus species. Genomic Xstir populations of X. borealis and X. laevis were mutually indistinguishable but significantly different from that of X. tropicalis. Received: 5 April 2000 / Accepted: 3 August 2000     

Characterization of Interrelated Sequence Motifs in Four Satellite DNAs and Their Distribution in the Genome of the Mollusc Donax trunculus

A family of four satellite DNAs has been characterized in the genome of the bivalve mollusc, Donax trunculus. All share HindIII sites, a similar monomer length of about 160 base pairs (bp), and the related oligonucleotide motifs GGTCA and GGGTTA, repeated six to 15 times within the repetitive units. The motif GGTCA is common to all members of the satellite family. It is present in three of them in both orientations, interspersed within nonrepetitive DNA sequences. The hexanucleotide GGGTTA appears to be the main building element of one of the satellites forming a prominent subrepeat structure in conjunction with the 5-bp motif. The former has been also found in perfect tandem repeats in a junction region adjacent to the proper satellite sequence. Southern analysis has revealed that (GGGTTA)_n and/or related sequences are abundant and widely distributed in the D. trunculus genome. The distribution observed is consistent with the concurrence of the scattering of short sequence motifs throughout the genome and the spread of longer DNA segments, with concomitant formation of satellite monomer repeats. Both kinds of dispersion may have contributed to the observed complex arrangement of the HindIII satellite DNA family in Donax. Received: 28 May 1996 / Accepted: 30 July 1996     

Recessive allelic variations of three microsatellite sites within the<Emphasis Type="Italic">O2</Emphasis> gene in maize

Recessive allelic variations were investigated at 3 microsatellite (SSR) sites within theO2 gene by using 14 inbredo2 lines and a wild-type line in maize. Among the 15 lines, allelic variations were observed at umc1066, phi057, and phi112 sites. Two alleles were found at the umc1066 site—a recessive allele with 2 perfect GCCAGA repeats and a dominant allele with 3 perfect repeats. Three alleles were found at the phi057 site—2 recessive alleles with 3 and 5 perfect GCC repeats, respectively, and another with 4 perfect repeats consistent with a dominant allele. At least 4 alleles exist at the phi112 site—among which 1 recessive allele has a 1-bp deletion, another has a 15-bp deletion, and other has no PCR products compared to the dominant allele; all the alleles have unchanged AG repeats. The phi057 site in exon 6 was identified to be a hypervariable region in the coding sequence of the02 gene, in addition to the 2 hypervariable regions in exon 1 previously reported. The primary mechanisms underlying the variations in repeat numbers and regions flanking the SSR within theO2 gene appear to be unequal crossing over and replication slippage. Furthermore, base substitution of SSR motif can create heteroalleles and modify the repeat number of SSR. The lysine content of kernel in theO2 ando2 lines correlates to a considerable extent with nucleotide variations at the umc1066, phi057, and phi112 sites. Our study suggests that it is best to use the 3 markers together in molecular marker-assisted selection for high-lysine maize materials.     

Absence of Protein Polymorphism in the Ras Genes of Drosophila melanogaster

Sequence analysis of 27 alleles of each of the three Ras-related genes in Drosophila melanogaster indicates that they all have low levels of polymorphism but may experience slightly different evolutionary pressures. No amino acid replacement substitutions were indicated in any of the sequences, or in the sibling species D. simulans and D. mauritiana. The Dras1 gene, which is the major ras homologue in Drosophila, has less within-species variation in D. melanogaster relative to the amount of divergence from the sibling species than does Dras2, although the contrast was not significant by the HKA test. Dras2 appears to be maintaining two classes of haplotype in D. melanogaster, one of which is closer to the alleles observed in the sibling species, suggesting that this is not likely to be a pseudogene despite the absence of a mutant phenotype. Although differences in level of expression may affect the function of the genes, it is concluded that genetic variation in the Ras signal transduction pathways cannot be attributed to catalytic variation in the Ras proteins. Received: 5 November 1998 / Accepted: 26 March 1999     

A Complex Organization of the Gene Encoding Cytochrome Oxidase Subunit 1 in the Mitochondrial Genome of the Dinoflagellate, Crypthecodinium cohnii: Homologous Recombination Generates Two Different cox1 Open Reading Frames

In the course of investigating mitochondrial genome organization in Crypthecodinium cohnii, a non-photosynthetic dinoflagellate, we identified four EcoRI fragments that hybridize to a probe specific for cox1, the gene that encodes subunit 1 of cytochrome oxidase. Cloning and sequence characterization of the four fragments (5.7, 5.1, 4.1, 3.5 kilobase pairs) revealed that cox1 exists in four distinct but related contexts in C. cohnii mtDNA, with a central repeat unit flanked by one of two possible upstream (flanking domain 1 or 2) and downstream (flanking domain 3 or 4) regions. The majority of the cox1 gene is located within the central repeat; however, the C-terminal portion of the open reading frame extends into flanking domains 3 and 4, thereby creating two distinct cox1 coding sequences. The 3′-terminal region of one of the cox1 reading frames can assume an elaborate secondary structure, which potentially could act to stabilize the mature mRNA against nucleolytic degradation. In addition, a high density of small inverted repeats (15–22 base pairs) has been identified at the 5′-end of cox1, further suggesting that hairpin structures could be important for gene regulation. The organization of cox1 in C. cohnii mtDNA appears to reflect homologous recombination events within the central repeat between different cox1 sequence contexts. Such recombining repeats are a characteristic feature of plant (angiosperm) mtDNA, but they have not previously been described in the mitochondrial genomes of protists. Received: 21 December 2000 / Accepted: 30 January 2001     

Evolution of the Larval Cuticle Proteins Coded by the Secondary Sex Chromosome Pair: X2 and Neo-Y of Drosophila miranda: I. Comparison at the DNA Sequence Level

The larval cuticle protein genes (Lcps) represent a multigene family located at the right arm of the metacentric autosome 2 (2R) in Drosophila melanogaster. Due to a chromosome fusion the Lcp locus of Drosophila miranda is situated on a pair of secondary sex chromosomes, the X2 and neo-Y chromosome. Comparing the DNA sequences from D. miranda and D. melanogaster organization and the gene arrangement of Lcp1–Lcp4 are similar, although the intergene distances vary considerably. The greatest difference between Lcp1 and Lcp2 is due to the occurrence of a pseudogene in D. melanogaster which is not present in D. miranda. Thus the cluster of the four Lcp genes existed already before the separation of the melanogaster and obscura group. Intraspecific homogenizations of different cluster units must have occurred repeatedly between the Lcp1/Lcp2 and Lcp3/Lcp4 sequence types. The most obvious example is exon 2 of the Lcp3 gene in D. miranda, which has been substituted by the corresponding section of the Lcp4 gene rather recently. The homogenization must have occurred before the translocation which generated the neo-Y chromosome. Lcp3 of D. melanogaster has therefore no orthologous partner in D. miranda. Rearrangements in the promoter regions of the D. miranda Lcp genes have generated new, potentially functional CAAT-box motifs. Since three of the Lcp alleles on the neo-Y are not expressed and Lcp3 is expressed only at a reduced level, it is suggestive to speculate that the rearrangements might be involved as cis-regulatory elements in the up-regulation of the X2-chromosomal Lcp alleles, in Drosophila an essential process for dosage compensation. The Lcp genes on the neo-Y chromosome have accumulated more base substitutions than the corresponding alleles on the X2. Received: 27 December 1995 / Accepted: 30 April 1996     

Sucrose Phosphate Synthase Genes in Plants Belong to Three Different Families

We present phylogenetic analyses to demonstrate that there are three families of sucrose phosphate synthase (SPS) genes present in higher plants. Two data sets were examined, one consisting of full-length proteins and a second larger set that covered a highly conserved region including the 14-3-3 binding region and the UDPGlu active site. Analysis of both datasets showed a well supported separation of known genes into three families, designated A, B, and C. The genomic sequences of Arabidopsis thaliana include a member in each family: two genes on chromosome 5 belong to Family A, one gene on chromosome 1 to Family B, and one gene on chromosome 4 to Family C. Each of three Citrus genes belong to one of the three families. Intron/exon organization of the four Arabidopsis genes differed according to phylogenetic analysis, with members of the same family from different species having similar genomic organization of their SPS genes. The two Family A genes on Arabidopsis chromosome 5 appear to be due to a recent duplication. Analysis of published literature and ESTs indicated that functional differentiation of the families was not obvious, although B family members appear not to be expressed in roots. B family genes were cloned from two Actinidia species and southern analysis indicated the presence of a single gene family, which contrasts to the multiple members of Family A in Actinidia. Only two family C genes have been reported to date. Received: 17 April 2001 / Accepted: 27 August 2001     

Distinct Annexin Subfamilies in Plants and Protists Diverged Prior to Animal Annexins and from a Common Ancestor

Annexin homologues in the kingdoms of Planta and Protista were characterized by molecular sequence analysis to determine their phylogenetic and structural relationship with annexins of Animalia. Sequence fragments from 19 plant annexins were identified in sequence databases and composite sequences were also assembled from expressed sequence tags for Arabidopsis thaliana. Length differences in protein amino-termini and evidence for unique exon splice sites indicated that plant annexins were distinct from those of animals. A third annexin gene of Giardia lamblia (Anx21-Gla) was identified as a distant relative to other protist annexins and to those of higher eukaryotes, thus providing a suitable outgroup for evolutionary reconstruction of the family tree. Rooted evolutionary trees portrayed protist, plant, and Dictyostelium annexins as early, monophyletic ramifications prior to the appearance of closely related animal annexin XIII. Molecular phylogenetic analyses of DNA and protein sequence alignments revealed at least seven separate plant subfamilies, represented by Anx18 (alfalfa, previously classified), Anx22 (thale cress), Anx23 (thale cress, cotton, rape and cabbage), Anx24 (bell pepper and tomato p34), Anx25 (strawberry, horseradish, pea, soybean, and castor bean), Anx26-Zma, and Anx27-Zma (maize). Other unique subfamilies may exist for rice, tomato p35, apple, and celery annexins. Consensus sequences compiled for each eukaryotic kingdom showed some breakdown of the ``annexin-fold' motif in repeats 2 and 3 of protist and plant annexins and a conserved codon deletion in repeat 3 of plants. The characterization of distinct annexin genes in plants and protists reflects their comparable diversity among animal species and offers alternative models for the comparative study of structure–function relationships within this important gene family. Received: 30 May 1996 / Accepted: 20 August 1996     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

Interelement Selection in the Regulatory Region of the copia Retrotransposon

We report the results of an analysis of naturally occurring cis-regulatory variation within and between two families of the copia Drosophila long terminal repeat (LTR) retrotransposon. The copia 5′ LTR and adjacent untranslated leader region (ULR) consists of a number of well-characterized sequence motifs which play a role in regulating expression of the element. In order to understand the evolutionary forces which may be responsible for generating and maintaining copia regulatory sequence variation, we have quantified levels of naturally occurring copia LTR-ULR nucleotide variation and subjected the data to a series of tests of neutrality. Our analysis indicates that the copia LTR-ULR has been subject to negative purifying selection within families and positive adaptive selection between families. We discuss these findings with respect to the regulatory evolution of retrotransposons and the phenomenon of interelement selection. Received: 5 February 1998 / Accepted: 14 May 1998     

Divergent Human Y-Chromosome Microsatellite Evolution Rates

In this work, we analyze several characteristics influencing the low variability of the microsatellite DYS19 in the major founder Amerindian Y chromosome lineage containing the point mutation DYS199-T. Variation of DYS19 was compared with that of five other Y-linked tetranucleotide repeat loci (DYS389A, DYS389B, DYS390, DYS391, and DYS393) in the DYS199-T lineage. All the other microsatellites showed significantly higher levels of variability than DYS19 as measured by gene diversity and repeat number variance. Moreover, we had previously shown that DYS19 had high diversity in Brazilians and in several other populations worldwide. Thus, the slow DYS19 evolution in the DYS199-T lineage seems to be both locus and allele specific. To understand the slow DYS19 evolutionary rate, the microsatellite loci were compared according to their mapping on the Y chromosome and also on the basis of structural aspects such as the base composition of the repeat motif and flanking regions and the degree of perfection and size (repeat number) of the variable blocks. The only observed difference that might be related to the low DYS19 variability is its small average number of repeats, a value expected to be closer to the founder DYS19 allele in the DYS199-T lineage. These data were also compared to other derived Y lineages. The Tat-C lineage displayed a lower DYS19 variability correlated to a small average repeat number, while in the DYS234-G lineage, a high DYS19 variability was found associated to a larger average repeat number. This approach reveals that evolution of Y microsatellites in lineages defined by slowly evolving markers, such as point mutations, can be greatly influenced by the size (number of repeats of the variable block) of the founder allele in each microsatellite locus. Thus lineage-dating methods using microsatellite variation should be practiced with great care. Received: 7 November 1998 / Accepted: 9 April 1999