Reduced Rates of Sequence Evolution of Y-Linked Satellite DNA in Rumex (Polygonaceae)

One characteristic of sex chromosomes is the accumulation of a set of different types of repetitive DNA sequences in the Y chromosomes. However, little is known about how this occurs or about how the absence of recombination affects the subsequent evolutionary fate of the repetitive sequences in the Y chromosome. Here we compare the evolutionary pathways leading to the appearance of three different families of satellite-DNA sequences within the genomes of Rumex acetosa and R. papillaris, two dioecious plant species with a complex XX/XY₁Y₂ sex-chromosome system. We have found that two of these families, one autosomic (the RAE730 family) and one Y-linked (the RAYSI family), arose independently from the ancestral duplication of the same 120-bp repeat unit. Conversely, a comparative analysis of the three satellite-DNA families reveals no evolutionary relationships between these two and the third, RAE180, also located in the Y chromosomes. However, we have demonstrated that, regardless of the mechanisms that gave rise to these families, satellite-DNA sequences have different evolutionary fates according to their location in different types of chromosomes. Specifically, those in the Y chromosomes have evolved at half the rate of those in the autosomes, our results supporting the hypothesis that satellite DNAs in nonrecombining Y chromosomes undergo lower rates of sequence evolution and homogenization than do satellite DNAs in autosomes.[Reviewing Editor: DR. Jerzy Jurka]     

Highly differentiated and conserved sex chromosome in fish species (Aulopus japonicus: Teleostei, Aulopidae)

While highly differentiated and long-conserved sex chromosomes such as XY and ZW chromosomes are observed, respectively, in mammalian and avian species, no counterparts to such chromosomes were observed in fish until we reported in the previous study that well-conserved and highly differentiated ZW sex chromosomes existed in the family of Synodontidae. Then, the problem was if the evolutionary history of the fish ZW chromosomes was long enough to be comparable to the mammalian and avian counterparts. To tackle the problem, we had to extend our finding of the fish sex chromosomes further than a family alone. For this purpose, we chose Aulopus japonicus that belonged to one of the related families to Synodontidae.Our cytogenetic and fluorescence in situ hybridization (FISH) analyses have clearly demonstrated that A. japonicus also has ZW chromosomes. We have also found that 5S rDNA clusters are located on the Z and W chromosomes in this species. Using nontranscribed intergenic sequences in the 5S rDNA clusters as PCR primers, we successfully amplified a 6-kb-long female-specific sequence on the W chromosome. The 6-kb-long sequence contained one transposable element and two tRNA sequences. The function of the sequence remains to be studied. Our Southern blot analysis confirmed that the 6-kb sequence was located only on the W chromosome.Therefore, it is now said that highly differentiated ZW chromosomes have been conserved over two fish families. As these families were reported to have been diverged 30-60 million years ago, the fish ZW chromosomes have an evolutionary history corresponding to the history of the families. This is perhaps the first case that fish sex chromosomes are shown to have such a long evolutionary lineage.     

Satellite DNA content illuminates the ancestry of a supernumerary (B) chromosome

B chromosomes are supernumerary genomic elements most likely derived from the standard (A) chromosomes, whose dispensability has freed their DNA sequences to evolve fast, thus making it difficult to uncover their ancestry. Here, we show the ancestry of a B chromosome in the grasshopper Eumigus monticola by means of the high-throughput analysis of the satellitome, i.e., the whole collection of satellite DNA (satDNA). The satellitome found in this species consists of 27 satDNA families, with monomer length between 5 and 325 nt and A + T content between 42.9 and 83.3 %. Two out of the 20 clustered satDNA families (EmoSat26–41 and EmoSat27–102) were observed only on the B chromosome. The A chromosome carrying the highest number of satDNA families was the megameric S8 (13 families), six of which were also present in the B chromosome, and three of these were exclusive of the S8 and B chromosomes. The absence in the B chromosome of the H3 histone gene cluster (located interstitially on S8) and three satDNA families (located distally on S8) allowed delimiting the possible origin of the B chromosome to the proximal third of the S8 autosome, through a breakpoint between EmoSat11–122 and the H3 cluster. Interestingly, bioinformatic analysis revealed the presence of seeds for the two B-specific satDNAs in the A chromosomes, suggesting their massive amplification in the B chromosome after its origin. Therefore, intraspecifically arisen B chromosomes can harbor DNA sequences apparently being B-specific.     

Centromeric repetitive DNA sequences in the genus Brassica

Representatives of two major repetitive DNA sequence families from the diploid Brassica species B. campestris and B. oleracea were isolated, sequenced and localized to chromosomes by in situ hybridization. Both sequences were located near the centromeres of many chromosome pairs in both diploid species, but major sites of the two probes were all on different chromosome pairs. Such chromosome specificity is unusual for plant paracentromeric repetitive DNA. Reduction of stringency of hybridization gave centromeric hybridization sites on more chromosomes, indicating that there are divergent sequences present on other chromosomes. In tetraploid species derived from the diploids, the number of hybridization sites was different from the sum of the diploid ancestors, and some chromosomes had both sequences, indicating relatively rapid homogenization and copy number evolution since the origin of the tetraploid species.     

Isolation and characterization of two families of satellite DNA with repetitive units of 135 bp and 2.5 kb in the ant Monomorium subopacum (Hymenoptera, Formicidae)

Analyzing the satellite DNA in the ant species Monomorium subopacum we found two unrelated families of satellite DNA. Because these satellite DNA families were isolated using the two enzymes HaeIII and EcoRI we called the two families HaeIII and EcoRI family, respectively. The HaeIII family proved to be organized in a 135-bp basic unit repeat, the EcoRI family in a 2.5-kb basic unit repeat. The latter represents perhaps the longest satellite DNA isolated up to now in insects. The HaeIII family apparently comprises about 10% of the total genomic DNA whereas the EcoRI family represents only about 1-2%. A comparative analysis of the two satellite DNA sequences showed no homology between the two families although both sequences possessed long A and T stretches. Eight of the 34 chromosomes showed hybridization with the HaeIII family and hybridization signals are visible in six chromosomes with the EcoRI family. Analysis of the electrophoretic mobility of satellite DNA on non-denaturing polyacrylamide showed that the HaeIII family is only slightly curved. However, the unit of the EcoRI satellite DNA family has curvature, especially the first 1000 bp of the monomeric repeat, in which this DNA is AT rich and has numerous A and T stretches. There are also internal inverted subrepeats in each family. The sequences of satellite DNA families found in Monomorium subopacum are different from the sequences of other satellite DNAs cloned in insects, including other species of ants.     

Centromere-specific repetitive sequences from Torenia, a model plant for interspecific fertilization, and whole-mount FISH of its interspecific hybrid embryos

Torenia fournieri is a good model plant to study fertilization in plants because it is easy to observe the fertilization process due to the protruding nature of the embryo sac. To study the location and movement of chromosomes and their centromeres in early embryogenesis of interspecific hybrid plants, we isolated two families of centromere-specific tandem repetitive sequences from T. fournieri and T. bailonii, and named them the "TCEN-family" and "BCEN-family", respectively. Both sequences consisted of a repeat unit of 52 bp located in the pericentric and centric heterochromatins. All signals of both sequences were prominent, but their intensity varied among the chromosomes. DNA-blot hybridization indicated the presence of similar sequences of TCEN-family in T. concolor, N. caerulea, and "Summer Wave", whereas the BCEN-family was found only in T. bailonii, thus indicating the wide or specific distribution of their repetitive families observed. We also applied whole-mount FISH to the interspecific hybrid embryos by using TCEN- and BCEN-family sequences as probes. Our results suggest that whole-mount FISH with the species-specific centromere sequences as probes is an ideal method to analyze the dynamics of chromosomes and centromeres in interspecific fertilization and early embryogenesis.     

A cloned sequence,p82H,of the alphoid repeated DNA family found at the centromeres of all human chromosomes

Clone p82H is a human DNA sequence which hybridises in situ exclusively to the centromeric regions of all human chromosomes. It is composed of approximately 14 tandemly repeated variants of a basic 172 bp sequence, and is related to the alphoid family. The organisation of the family of cross-hybridising sequences, detected by the clone p82H, is described both in the human genome and on certain chromosomes, and its relationship to known sequence families is discussed.     

A molecular deletion of distal chromosome 4p in two families with a satellited chromosome 4 lacking the Wolf-Hirschhorn syndrome phenotype.

We report two families with a satellited chromosome 4 short arm (4ps). Satellites and stalks normally occur on the short arms of acrocentric chromosomes; however, the literature cites several reports of satellited nonacrocentric chromosomes, which presumably result from a translocation with an acrocentric chromosome. This is the first report of 4ps chromosomes. Our families are remarkable in that both unaffected and affected individuals carry the 4ps chromosome. The phenotypes observed in affected individuals, although dissimilar, were sufficient to encourage a search for a deletion of chromosome 4p. By Southern blot analysis and fluorescence in situ hybridization, a deletion of material mapping approximately 150 kb from chromosome 4pter was discovered. This deletion is notable because it does not result in the Wolf-Hirschhorn syndrome and can result in an apparently normal phenotype. We speculate that homology between subterminal repeat sequences on 4p and sequences on the acrocentric short arms may explain the origin of the rearrangement and that position effect may play a role in the expression of the abnormal phenotype.     

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C₀t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes.     

Germ line-specific DNA sequences are present on all five micronuclear chromosomes in Tetrahymena thermophila.

The development of the macronucleus from the zygotic micronucleus in the ciliated protozoan Tetrahymena spp. involves the elimination of specific DNA sequences (M. C. Yao and M. Gorovsky, Chromosoma 48:1-18 1974). The present study demonstrates that micronucleus-specific DNA is present on all five of the micronuclear chromosomes. Fragments of micronuclear DNA from Tetrahymena thermophila were cloned in the plasmid vector pBR322. A procedure was developed to examine the organization of the cloned sequences in micro- and macronuclear DNA without nick translating each individual probe. Twenty-three percent of randomly selected DNA sequences examined by this method were micronucleus (germ line) specific. They were all members of families of repeated sequences. Hybridization of six micronucleus-specific DNA sequences to micronuclear DNA from nullisomic strains of T. thermophila, which are lacking one or more pairs of chromosomes in the micronucleus, suggested that these sequences are present on several chromosomes. One micronucleus-specific sequence was shown by in situ hybridization to be present on all five of the micronuclear chromosomes.     

Different patterns in molecular evolution of the Triticeae

A huge part of the genomes of most Triticeae species is formed by different families of repetitive DNA sequences. In this paper the phylogenetic distribution of two major classes of the repeats, retrotransposons and tandemly organized DNA sequences, are considered and compared with the evolution of gene-rich regions and generally accepted Triticeae phylogenetic relationships. In Hordeum, LTR-containing retrotransposons are dispersed along the chromosomes and are consistent with the existing picture of the phylogeny of Hordeum. Another retrotransposon class, LINEs, have evolved independently from LTR-retrotransposons. Different retrotransposon classes appear to have competed for genome space during the evolution of Hordeum. Another class of repeats, tandemly organized DNA sequences, tends to cluster at the functionally important regions of chromosomes, centromeres and telomeres. The distribution of a number of tandem DNA families in Triticeae is not congruent with generally accepted phylogenetic relationships. While natural selection is the dominant factor determining the structure of genic regions we suggest that the contribution of random events is important in the evolution of repetitive DNA sequences. The interplay of stochastic processes, molecular drive, and selection determines the structure of chromosomal regions, notably at centromeres and telomeres, stabilizing and differentiating species-specific karyotypes. Thus, the evolution of these regions may occur largely independently of the evolution of gene-rich regions.     

The genomics of long tandem arrays of satellite DNA in the human genome

At least 10% of DNA in the human genome consists of long arrays of repeated sequences, arranged in tandem head-to-tail arrays in a number of discrete, highly localized chromosomal regions. Different families of these so-called "satellite DNA" sequences have been defined, organized in diverged subsets on different chromosomes. The molecular, cytogenetic, and evolutionary analysis of the hierarchical organization of such sequences in the human and other complex genomes encompasses a variety of approaches, including chromosomal mapping, in situ hybridization, genetic linkage analysis, long-range restriction mapping, and DNA sequencing. Investigation of the organization of satellite arrays constitutes a necessary first step towards eventual elucidation of the origin, evolution, and maintenance of these sequences and their contribution to the structure and behavior of human chromosomes.     

Multiple alpha and beta tubulin genes represent unlinked and dispersed gene families

Cloned cDNA sequences specific for alpha or beta tubulin mRNAs have been used to show that the multigene families which encode either alpha or beta tubulin are unlinked and dispersed throughout the chicken genome. Fractions of chicken chromosomes partially purified by centrifugation on a sucrose gradient were digested with restriction endonucleases and electrophoresed on agarose gels. The DNA was transferred to nitrocellulose filters and hybridized to labeled probes constructed from cloned cDNA sequences specific for alpha or beta tubulin. We find alpha tubulin sequences on four different chicken chromosomes and beta tubulin sequences on at least two different chromosomes. Moreover, using chicken chromosomes further purified with a fluorescent cell sorter, we have been able unambiguously to localize alpha tubulin genes to chromosome 1 and chromosome 8 and two of the beta genes to chromosome 2.     

Identification of DNA sequences flanking the breakpoint of human t(14q21q) Robertsonian translocations.

We have employed molecular probes and in situ hybridization to investigate the DNA sequences flanking the breakpoint of a group of t(14q21q) Robertsonian translocations. In all the families studied, the probands were patients with Down syndrome who carried a de novo t(14q21q) translocation. The DNA probes used were two alphoid sequences, alphaRI and alphaXT, which are specific for the centromeres of chromosomes 13 and 21 and of chromosomes 14 and 22, respectively; a satellite III sequence, pTRS-47, which is specific for the proximal p11 region of chromosomes 14 and 22; and a newly defined satellite III DNA, pTRS-63, which is specific for the distal p11 region of chromosome 14. The two alphoid probes detected approximately the same amount of autoradiographic signal on the translocated chromosomes as was expected for chromosomes 14 and 21 of the originating parent, suggesting that there has been no loss of these centromeric sequences during the translocation events. Results with the two satellite III probes indicated that the domain corresponding to pTRS-47 was retained in the translocated chromosomes, whereas the domain for pTRS-63 was lost. These results have allowed us to place the translocation breakpoint between the pTRS-47 and pTRS-63 domains within the p11 region of chromosome 14.     

Structural organization and polymorphism of the alpha satellite DNA sequences of chromosomes 13 and 21 as revealed by pulse field gel electrophoresis

Summary More than 30 unrelated individuals were analysed by pulse field gel electrophoresis for the alphoid centromeric sequences of chromosomes 13 and 21. These individuals had DNA patterns all different from each other and were most probably heterozygous at both loci. When several nuclear families were analysed in this manner, segregation was shown to be Mendelian, and no recombination event was detected over the 150 meioses scored in this study. Alphoid DNA sequences, therefore, constitute highly polymorphic centromeric markers, which can be used in linkage analysis for loci close to the centromeres of chromosomes 13 and 21.     

Dispersion of argininosuccinate synthetase-like human genes to multiple autosomes and the X chromosome

DNA sequences closely homologous to argininosuccinate synthetase are present at ten or more distinct locations in the human genome, including sites on chromosomes 6,9 and X. Argininosuccinate synthetase thus represents one of the most widely dispersed multigene families described to date, the first instance of a multigene family associated with an enzyme of intermediary metabolism and, perhaps most striking, the first instance of a multigene family with members on both autosomes and sex chromosomes.     

Isolation of CAG/CTG repeats from within the chromosome 2p21-p24 locus for autosomal dominant spastic paraplegia (SPG4) by YAC fragmentation

Pure autosomal dominant spastic paraplegia (SPG) is a genetically heterogeneous neurodegenerative disorder of the central nervous system clinically characterized by progressive spasticity mainly affecting the lower limbs. Three distinct loci have been mapped to chromosomes 14q (SPG3), 2p (SPG4) and 15q (SPG6). In particular, SPG4 families show striking intrafamilial variability suggestive of anticipation and evidence has been provided that CAG/CTG repeat expansions may be involved. To isolate CAG/CTG repeat containing sequences from within the SPG4 candidate region, a novel approach was developed. Fragmentation vectors were assembled allowing direct fragmentation of yeast artificial chromosomes (YACs) with a short (> or = 21 bp) CAG/CTG sequence as the target site for homologous recombination. We used the CAG/CTG YAC fragmentation vectors to isolate CAG/CTG containing sequences from four YACs spanning the SPG4 candidate region between D2S400 and D2S367. A total of four CAG/CTG containing sequences were isolated of which three were novel. However, none of the four CAG/CTG repeats showed expanded alleles in two Belgian SPG4 families. In addition, we showed that the CAG/CTG alleles detected by the repeat expansion detection (RED) method could be fully explained by two polymorphic nonpathogenic CAG/CTG repeats on chromosomes 17 and 18, respectively. Also, the RED expansions in six SPG families could not be explained by amplification of the CAG/CTG repeats at the SPG4 locus. Together, our data do not support the hypothesis of a CAG/CTG repeat expansion as the molecular mechanism underlying SPG4 pathology.     

Concerted evolution of members of the multisequence family chAB4 located on various nonhomologous chromosomes

During the last years it became obvious that a lot of families of long-range repetitive DNA elements are located within the genomes of mammals. The principles underlying the evolution of such families, therefore, may have a greater impact than anticipated on the evolution of the mammalian genome as a whole. One of these families, called chAB4, is represented with about 50 copies within the human and the chimpanzee genomes and with only a few copies in the genomes of gorilla, orang-utan, and gibbon. Members of chAB4 are located on 10 different human chromosomes. FISH of chAB4-specific probes to chromosome preparations of the great apes showed that chAB4 is located, with only one exception, at orthologous places in the human and the chimpanzee genome. About half the copies in the human genome belong to two species-specific subfamilies that evolved after the divergence of the human and the chimpanzee lineages. The analysis of chAB4-specific PCR-products derived from DNA of rodent/human cell hybrids showed that members of the two human-specific subfamilies can be found on 9 of the 10 chAB4-carrying chromosomes. Taken together, these results demonstrate that the members of DNA sequence families can evolve as a unit despite their location at multiple sites on different chromosomes. The concerted evolution of the family members is a result of frequent exchanges of DNA sequences between copies located on different chromosomes. Interchromosomal exchanges apparently take place without greater alterations in chromosome structure. Received: 20 March 1997 / Accepted: 13 September 1997     

Sequence replication and banding organization in the polytene chromosomes of Drosophila melanogaster

The relative proportions of cloned DNA fragments from all known hierarchies of sequence organization in polytene and diploid chromosomes were compared. It was found that unique sequences of varying sizes and chromosomal locations are equally replicated in salivary gland chromosomes. Sequences of euchromatic polydisperse gene families are also replicated proportionately in polytene and diploid tissues. Perhaps the most significant finding is that the histone gene repeats, despite their normal banding organization, are under-replicated in the polytene chromosome of Drosophila melanogaster. However, the clustered and well-banded 5S genes are most likely equally replicated. It is therefore concluded that differential sequence replication plays no apparent role in either the assembly or morphology of a band; and likewise, the assembly of polytenic DNA into band units is not affected by either the local abundancy or arrangement of middle repetitive sequences. The likelihood that the clustered arrangement is an important factor in the selection of sequences for under-replication is discussed.