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.     

Primate evolution of a dispersed human repetitive DNA sequence

A dispersed middle repetitive DNA sequence isolated originally from human chromosome 12 did not show homology with rodent DNA under standard conditions of Southern DNA blot analysis. The evolutionary relationship of this human repetitive DNA to that of other primates was investigated using three hybridization methods: DNA dot blot, Southern DNA blot analysis, and chromosome in situ hybridization. Homology with the human repetitive DNA was found throughout the suborder Anthropoidea, in fourteen ape and New and Old World monkey species. In addition, the human pattern of hybridization to noncentromeric regions of all chromosomes was seen. No hybridization by any of the three techniques was found in five species of the suborder Prosimii. The phenomenon of marked differences in sequence homology and copy number of dispersed repetitive DNA from closely related species has been observed in protozoans (Plasmodia), Drosophila, sea urchins, mice and the great apes (Hominoidea). We report here a similar phenomenon that may have occurred at an early stage in primate evolution.     

Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences

A set of six cloned barley (Hordeum vulgare) repetitive DNA sequences was used for the analysis of phylogenetic relationships among 31 species (46 taxa) of the genus Hordeum, using molecular hybridization techniques. in situ hybridization experiments showed dispersed organization of the sequences over all chromosomes of H. vulgare and the wild barley species H. bulbosum, H. marinum and H. murinum. Southern blot hybridization revealed different levels of polymorphism among barley species and the RFLP data were used to generate a phylogenetic tree for the genus Hordeum. Our data are in a good agreement with the classification system which suggests the division of the genus into four major groups, containing the genomes I, X, Y, and H. However, our investigation also supports previous molecular studies of barley species where the unique position of H. bulbosum has been pointed out. In our experiments, H. bulbosum generally had hybridization patterns different from those of H. vulgare, although both carry the I genome. Based on our results we present a hypothesis concerning the possible origin and phylogeny of the polyploid barley species H. secalinum, H. depressum and the H. brachyantherum complex.     

The distribution, organization and evolution of two abundant and widespread repetitive DNA sequences in the genus Hordeum

The genomic organization and chromosomal distributions of two abundant tandemly repeated DNA sequences, dpTa1 and pSc119.2, were examined in six wild Hordeum taxa, representing the four basic genomes of the genus, by Southern and fluorescence in situ hybridization. The dpTa1 probe hybridized to between 30 and 60 sites on the chromosomes of all five diploid species studied, but hybridization patterns differed among the species. Hybridization of the pSc119.2 sequence to the chromosomes and Southern blots of digested DNA detected signals in Hordeum bulbosum, Hordeum chilense, Hordeum marinum and Hordeum murinum 4x, but not in Hordeum murinum 2x and Hordeum vulgare ssp. spontaneum. A maximum of one pSc119.2 signal was observed in the terminal or subterminal region of each chromosome arm in the species carrying this sequence. The species carrying the same I-genome differed in the presence (Hordeum bulbosum) or absence (Hordeum spontaneum) of pSc119.2. The presence of pSc119.2 in the tetraploid cytotype of Hordeum murinum, but its absence in the diploid cytotype, suggests that the tetraploid is not likely to be a simple autotetraploid of the diploid. Data about the inter- and intra-specific variation of the two independent repetitive DNA sequences give information about both the interrelationships of the species and the evolution of the repetitive sequences. Received: 17 March 1999 / Accepted: 16 June 1999     

Characterization of four dispersed repetitive DNA sequences from Zea mays and their use in constructing contiguous DNA fragments using YAC clones.

We have isolated four repetitive DNA fragments from maize DNA. Only one of these sequences showed homology to sequences within the EMBL database, despite each having an estimated copy number of between 3 x 104 and 5 x 104 per haploid genome. Hybridization of the four repeats to maize mitotic chromosomes showed that the sequences are evenly dispersed throughout most, but not all, of the maize genome, whereas hybridization to yeast colonies containing random maize DNA fragments inserted into yeast artificial chromosomes (YACs) indicated that there was considerable clustering of the repeats at a local level. We have exploited the distribution of the repeats to produce repetitive sequence fingerprints of individual YAC clones. These fingerprints not only provide information about the occurrence and organization of the repetitive sequences within the maize genome, but they can also be used to determine the organization of overlapping maize YAC clones within a contiguous fragment (contigs). Key words : maize, repetitive DNA, YACs.     

Genome-specific repetitive sequences in the genus Oryza

Summary Repetitive DNA sequences are useful molecular markers for studying plant genome evolution and species divergence. In this paper, we report the isolation and characterization of four genome-type specific repetitive DNA sequences in the genus Oryza. Sequences specific to the AA, CC, EE or FF genome types are described. These genome-type specific repetitive sequences will be useful in classifying unknown species of wild or domestic rice, and in studying genome evolution at the molecular level. Using an AA genome-specific repetitive DNA sequence (pOs48) as a hybridization probe, considerable differences in its copy number were found among different varieties of Asian-cultivated rice (O. sativa) and other related species within the AA genome type. Thus, the relationship among some of the members of AA genome type can be deduced based on the degree of DNA sequence similarity of this repetitive sequence.     

Variation in nuclear DNA in the genus Secale

Estimates of the 4C DNA amount per nucleus in 16 taxa of the genus Secale made by Feulgen microdensitometry ranged from 28.85 picograms (pg) in S. silvestre PBI R52 to 34.58 pg in S. vavilovii UM 2D49, compared with 33.14 pg in S. cereale cv. Petkus Spring which was used as a standard. Giemsa C-banding patterns showed considerable interspecific and intraspecific variation and several instances of polymorphism for large telomeric C-bands. The proportion of telomeric heterochromatin in the genome ranged from about 6% in S. silvestre and S. africanum to about 12% in cultivated rye. A detailed comparison of nine taxa showed no overall relationship between 4C DNA amount and the proportion of telomeric heterochromatin in the genome. However, evidence is presented which strongly supports the notion that the major evolutionary change in chromosome structure in Secale has involved the addition of heterochromatin at, or close to, the telomeres. It is suggested that saltatory amplification events at telomeres were initially responsible for each large increase in DNA amount. Subsequently unequal crossing over between homologues may have played an important secondary role by extending the range of variation in the amount of heterochromatin at a given telomere, while crossing over between non-homologues may have provided a useful mechanism allowing an increase in the DNA amount at one telomere to be distributed between chromosomes.     

The evolution of repetitive DNA sequences in sea urchins.

Molecular hybridization of nuclear DNAs has been employed to study the evolution of the repetitive DNA sequences in four species of sea urchin. The data show that relative to S. purpuratus there has been approximately 0.1% sequence divergence per million years in the repetitive DNA sequences of S. droebachiensis, S. franciscanus, and L. pictus. These results confirm that repetitive DNA sequences are strongly conserved during evolution. However, comparison of the extent of base pair mismatch in the repetitive DNA heteroduplexes formed at Cot 20 with those formed at Cot 200 during the hybridization of S. purpuratus and L. pictus DNAs reveals that highly repetitive sequences of sea urchins may diverge more rapidly than do the more moderately repetitive sequences.     

Cytological localization and organization of dispersed middle repetitive DNA sequences of Drosophila subobscura

To study the middle repetitive fraction of the Drosophila subobscura genome, 26 phage clones containing repetitive sequences were examined by Southern DNA blot analysis and by in situ hybridization to polytene chromosomes. These results led to a classification of the clones according to five different types of hybridization patterns. Two types, each containing seven clones, are characterized by hybridization at 100 to 300 sites dispersed over the euchromatic parts of the chromosomes, and in addition by one prominently labelled chromosome band. One of these two classes also showed strong labelling of the chromocentre. The remaining types of hybridization pattern lacked a prominent band but showed hybridization either to the euchromatic regions or to the chromocentre or both. Chromosome A (=X) was the preferred location of prominently labelled bands and it also showed an excess of labelling by some clones. Some of the cloned dispersed sequences were localized cytologically on chromosomes of larvae from crosses between different strains of D. subobscura and between two closely related species, in order to detect heterozygosity at hybridization sites. Comparisons of the chromosomal distribution of labelling sites showed differences in number and location, indicating the possibility of transposition events.     

Evolutionary trends of different repetitive DNA sequences during speciation in the genus secale

The presence and distribution of two simple sequence repeats (SSRs), three highly repetitive sequences from rye, and the 5S rDNA have been investigated in 3 rye cultivars and 10 wild-related species of the genus SECALE: The following conclusions can be drawn in addition to detailed knowledge of the sequence content of chromatin in each accession studied: (1) Every species is unique in either or both the complement and chromosomal distribution of the six repeated sequences analyzed. (2) These sequences reveal multiple landmarks along all the rye chromosomes arms. (3) High polymorphism as well as heterozygosity between homologues in the distribution of the (AAG)(5) and (AAC)(5) was revealed in the outbreeding species of the Secale strictum complex. (4) It is possible to deduce trends in the complexity of repetitive DNA during the evolution of the genus. A possible evolutionary pathway that accounts for the present-day Secale species is presented.     

The structure,amount and chromosomal localisation of defined repeated DNA sequences in species of the genus Secale

The structure, copy number and chromosomal location of arrays of four families of highly repeated sequences have been investigated in representative species of the genus Secale. The four unrelated families, previously characterised in Secale cereale, have repeating units of 480, 610, 630 and 120 base pairs respectively. The following general conclusions can be drawn in addition to detailed knowledge of the sequence content of heterochromatin in each accession studied: (1) Every species is unique in its complement or chromosomal distribution or both of the four highly repeated sequence families. S. montanum and S. cereale accessions studied here show the same complement of repeated sequences, but they differ substantially in the amounts they contain of the 610 and 630 base pair (bp) families, and in the distribution over the chromosomes of the 480 bp family. The structure of the repeating unit is also different in many members of the 480 bp family in S. montanum. — (2) The substantial differences between species in the amounts of the most highly repeated DNA sequences exist in the absence of any such conspicuous differences in most other repeated sequences which were detected as fluorescent bands after restriction enzyme digestion and gel electrophoresis. — (3) Each of the different highly repeated families can exist independently of the other families, though all the families have telomeric sites. Also, in the outbreeding species, heteromorphisms are frequent, and are particularly conspicuous in hybridisation detecting the 480 bp sequence family. — (4) The association of the highly repeated sequences with heterochromatin, discussed in the accompanying paper is generally true for other species in the genus, and the lower amounts of heterochromatin in other Secale species compared to S. cereale are associated with lower amounts of specific families of highly repeated DNA sequences. — (5) Analysis of highly repeated sequence families is likely to provide an easy method of identification of new accessions of Secale.     

Coordinate replication of dispersed repetitive sequences in Physarum polycephalum

The synchronous macroplasmodial growth phase of the slime mould Physarum polycephalum was used to study the in vivo replication of large chromosomal DNA segments. Newly replicated DNA was isolated at various points in S-phase by its preferential association with the nuclear matrix. This DNA was then used to probe cosmid clones of the Physarum genome. The results indicate that certain dispersed repetitive sequences in the genome are coordinately replicated. The observed pattern of replication may be due either to the presence of a replication origin within each repetitive sequence or to the systematic arrangement of these sequences around a replication origin. The latter appears more likely since the repetitive sequences are probably not randomly scattered within the genome.     

Sequence variation in dispersed repetitive sequences in Saccharomyces cerevisiae

Two new dispersed repetitive DNA sequences related to the transposable element Tyl have been isolated from the genome of Saccharomyces cerevisiae. One sequence, designated Tyl-17, is present at about six copies per haploid genome, and one copy is located approximately 1000 base-pairs from the LEU2 locus on chromosome III. Tyl-17 is about the same size as Tyl (Cameron et al., 1979) and is flanked by δ sequences, but differs from Tyl by the presence of two large substitutions representing about 50% of the sequence. Tyl and Tyl-17 are found in a ‘head-to-head’ array in at least one cloned region of the yeast genome. Another sequence, designated Tyl-161, is situated about 9000 base-pairs from the PGK locus of chromosome III, and is structurally identical to Tyl except for the presence of a 1200 base-pair insertion near one end of the sequence element.     

The evolution of the long and short repetitive DNA sequences in sea urchins.

The rates of evolution of purified long and short repetitive DNA sequences were examined by hybridisation analysis between the DNAs from several species of sea urchins. We find that the rates of nucleotide substitution are very comparable within mutually retained sequences for the two classes of repetitive DNA. The loss of hybridisable sequences between species also occurs at similar rates among both the short and long repetitive DNA sequences. Between species that separated less than 50 million years ago, hybridisable short repetitive sequences are lost all through the spectrum of reiteration frequencies. The long repeats contain a few sequences which are highly conserved within all of the species examined, and which amount to approximately 1% of the total genome. The short repetitive class, on the other hand, does not seem to contain any such highly conserved elements. The long repetitive sequences internally appear to contain short 'units' of reiteration, which may comprise families within the long repetitive class. We find no evidence to indicate that the majority of long and short repetitive sequences evolve by different mechanisms or at different rates.     

Chromosomes,DNA sequences,and evolution in salamanders of the genus Plethodon

Chromosomes and DNA sequence homologies have been studied in 15 species of North American salamander belonging to the genus Plethodon. These include 4 Eastern small species, 5 Eastern large species, 5 Western, and 1 New Mexican species. All species have 14 metacentric or sub-metacentric chromosomes. Their karyotypes are closely similar, but their C values range from 18–69 pg. DNA:DNA molecular hybridization studies showed that salamanders belonging to the same species group had between 60 and 90% of the observed repetitive DNA sequences in common, different groups of Eastern species had between 40 and 60% in common, and Eastern and Western groups had less than 10% in common. The slowly reassociating DNA sequences were also diverse among species, but higher levels of homology were observed than in the case of repetitive sequences. The New Mexican species was exceptional in showing little homology with other species with respect to either repetitive or slowly reassociating sequences.     

Chromosomes,DNA sequences,and evolution in salamanders of the genus Aneides

Chromosomes and DNA sequence homologies have been studied in salamanders of the genus Aneides. The species studied included A. ferreus, flavipunctatus, lugubris, hardii and aeneus. All species have 14 chromosomes. The karyotypes of A. ferreus and A. hardii are very similar. All chromosomes are metacentric or sub-metacentric except chromosome 13 which is telocentric in A. hardii, but is represented by a telocentric and a sub-telocentric chromosome in A. ferreus. C values range from 35.2 to 46.0 pg. Salamanders from different species groups have nothing in common with respect to that fraction of their repeated DNA sequences that hybridizes in experiments involving the binding of labelled whole complementary RNA from one species to whole DNA from another species. Salamanders from the same species group (ferreus, lugubris and flavipunctatus) have about 25% in common with respect to their repetitive DNA sequences.     

Simple DNA sequences and dispersed repetitive elements in the vicinity of mouse immunoglobulin K light chain genes

The simple DNA sequences (T-G)20, (T-T-T-G-C)20 and (G-C-C-T-C-T)30 were found in the vicinity of mouse immunoglobulin genes and of dispersed repetitive elements as the R, B1 and B2 sequences. On the basis of sequence data, blot hybridizations with salmon and mouse DNA and with defined mouse DNA fragments, possible functional and evolutionary aspects of simple DNA sequences are discussed.     

Length and interspersion of repetitive and non repetitive DNA sequences in four Amphibian species with different genome sizes

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.