Two Tandemly Arrayed Transfer-RNA-Derived SINEs of the Medaka (<Emphasis Type="BoldItalic">Oryzias latipes</Emphasis>)

: Two tandemly arrayed short interspersed repetitive element (SINE) sequences were found in medaka (Oryzias latipes). These two SINE sequences, designated SINE1 and SINE2, were flanked by a 180-bp AT-rich region. Both appeared to be derived from transfer RNA. The former exhibited 80% sequence homology to human tRNA^Ala and the latter exhibited 94% sequence homology to rat tRNA^Ser. SINE1 contained the retroviral U5 region, whereas SINE2 did not. This is the first sequence-level demonstration of the existence of neighboring SINEs in medaka.     

Analysis of rat repetitive DNA sequences.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

Molecular characterization and evolution of an interspersed repetitive DNA family of oysters

When genomic DNA from the European flat oyster Ostrea edulis L. was digested by BclI enzyme, a band of about 150?bp was observed in agarose gel. After cloning and sequencing this band and analysing their molecular characteristics and genomic organization by means of Southern blot, in situ hybridisation, and polymerase chain reaction (PCR) protocols, we concluded that this band is an interspersed highly repeated DNA element, which is related in sequence to the flanking regions of (CT)-microsatellite loci of the species O. edulis and Crassostrea gigas. Furthermore, we determined that this element forms part of a longer repetitive unit of 268?bp in length that, at least in some loci, is present in more than one copy. By Southern blot hybridisation and PCR amplifications-using primers designed for conserved regions of the 150-bp BclI clones of O. edulis-we determined that this repetitive DNA family is conserved in five other oyster species (O. stentina, C. angulata, C. gigas, C. ariakensis, and C. sikamea) while it is apparently absent in C. gasar. Finally, based on the analysis of the repetitive units in these oyster species, we discuss the slow degree of concerted evolution in this interspersed repetitive DNA family and its use for phylogenetic analysis.     

A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

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.     

A detailed linkage map of medaka, Oryzias latipes: comparative genomics and genome evolution

We mapped 633 markers (488 AFLPs, 28 RAPDs, 34 IRSs, 75 ESTs, 4 STSs, and 4 phenotypic markers) for the Medaka Oryzias latipes, a teleost fish of the order Beloniformes. Linkage was determined using a reference typing DNA panel from 39 cell lines derived from backcross progeny. This panel provided unlimited DNA for the accumulation of mapping data. The total map length of Medaka was 1354.5 cM and 24 linkage groups were detected, corresponding to the haploid chromosome number of the organism. Thirteen to 49 markers for each linkage group were obtained. Conserved synteny between Medaka and zebrafish was observed for 2 independent linkage groups. Unlike zebrafish, however, the Medaka linkage map showed obvious restriction of recombination on the linkage group containing the male-determining region (Y) locus compared to the autosomal chromosomes.     

Identification of the sex-determining locus in the Thai medaka, Oryzias minutillus

A sex-determining gene, DMY, which is comparable to the SRY gene in mammals, has been identified in the medaka, Oryzias latipes. Although Oryzias curvinotus, a closely related species to O. latipes also has DMY, this gene has not been found in other Oryzias fishes. It has recently been demonstrated that the sex chromosomes of Oryzias dancena and Oryzias hubbsi differ from those of O. latipes and these species have XX/XY and ZZ/ZW systems, respectively. This may suggest that Oryzias species have evolved different sex-determining genes on different sex chromosomes. In the present study, we investigated the sex determination mechanism in Oryzias minutillus, which is closely related to O. dancena and O. hubbsi. Linkage analysis using 14 isolated sex-linked DNA markers showed that this species has an XX/XY sex determination system. These sex-linked markers were located on linkage group 8 of O. latipes, suggesting that the sex chromosomes of O. minutillus are homologous to the autosomes of other Oryzias species. Furthermore, fluorescence in situ hybridization using a tightly sex-linked marker demonstrated that the XY sex chromosomes of O. minutillus and O. dancena were not homologous. These findings provide additional evidence for independent origins of sex chromosomes and sex-determining genes in these closely related species.     

The genome size evolution of medaka (Oryzias latipes) and fugu (Takifugu rubripes)

Evolution of the genome size in eukaryotes is often affected by changes in the noncoding sequences, for which insertions and deletions (indels) of small nucleotide sequences and amplification of repetitive elements are considered responsible. In this study, we compared the genomic DNA sequences of two kinds of fish, medaka (Oryzias latipes) and fugu (Takifugu rubripes), which show two-fold difference in the genome size (800 Mb vs. 400 Mb). We selected a contiguous DNA sequence of 790 kb from the medaka chromosome LG22 (linkage group 22), and made a precise comparison with the sequence (387 kb) of the corresponding region of Takifugu. The sequence of 178 kb in total was aligned common between two fishes, and the remaining sequences (612 kb for medaka and 209 kb for fugu) were found abundant in various repetitive elements including many types of unclassified low copy repeats, all of which accounted for more than a half (54%) of the genome size difference. Furthermore, we identified a significant difference in the length ratio of the unaligned sequences that locate between the aligned sequences (USBAS), particularly after eliminating known repetitive elements. These USBAS with no repetitive elements (USBAS-nr) located within the intron and intergenic region. These results strongly indicated that amplification of repetitive elements and compilation of indels are major driving forces to facilitate changes in the genome size.     

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.     

Analysis of major repetitive DNA sequences in the dog (Canis familiaris) genome

A systematic screening and analysis of repeated DNA sequences from a dog genomic library composed of small DNA inserts enabled us to characterize abundant canine repetitive DNA families. Four main families were identified: i) a group of highly repeated tRNA-derived short interspersed repetitive DNA elements (tRNA-SINEs); ii) another type of SINE-like element that was mainly found inserted into long interspersed repetitive elements (LINEs); iii) LINEs of the L1 type; and iv) satellite or satellite-like DNA. Surprisingly, no SINEs derived from 7SL RNA were found in the dog genome. These data should help in the analysis of canine DNA sequences and in the design of canine genome mapping reagents. Received: 4 November 1998 / Accepted: 2 February 1999     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

DNA sequence organization in the genomes of three related millet plant species

Summary A major portion of the genomes of three millet species, namely, barn yard millet, fox tail millet and little millet has been shown to consist of interspersed repeat and single copy DNA sequences. The interspersed repetitive DNA sequences are both short (0.15–1.0 kilo base pairs, 62–64% and long (>1.5 kilo base pairs, 36–38%) in barn yard millet and little millet while in fox tail millet, only long interspersed repeats (>1.5 kilo base pairs) are present. The length of the interspersed single copy DNA sequences varies in the range of 1.6–2.6 kilo base pairs in all the three species. The repetitive duplexes isolated after renaturation of 1.5 kilo base pairs and 20 kilo base pairs long DNA fragments exhibit a high thermal stability with Tms either equal to or greater than the corresponding native DNAs. The S1 nuclease resistant repetitive DNA duplexes also are thermally stable and reveal the presence of only 1–2% sequence divergence.The present data on the modes of sequence arrangement in millets substantiates the proposed trend in plants, namely, plants with 1C nuclear DNA content of less than 5 picograms have diverse patterns of sequence organization while those with 1C nuclear DNA content greater than 5 picograms have predominantly a short period interspersion pattern.Abbreviations kbp kilobase pairs NCL Communication No. 3606.     

The organization of the main component DNA of a crustacean genome with a paucity of middle repetitive sequences

The frequency classes and organization of the main component (mc) DNA of a crustacean, the land crab, Gecarcinus lateralis, have been characterized. The reassociation kinetics of 380 nucleotide long mcDNA fragments show that approximately 50% contain sequences repeated more than 800 times. Present in few, if any, copies are sequences repeated from 2 to 800 times. The remainder of the DNA reassociates as single copy sequences with a rate constant consistent with the organism's genome size. The reassociation kinetics of highly sheared DNA fragments of every true crab studied (Vaughn, 1975; Christie et al., 1976) are similar to each other and different from those of other invertebrate DNAs (Goldberg et al., 1975). Each of these genomes has a paucity of sequences repeated from 10 to 800 times and an abundance of highly repeated sequences. To determine if sequences repeated more than 800 times are interspersed with single copy sequences, we examined the arrangement of repetitive and non-repetitive sequences in mcDNA. The reassociation and melting properties of partially duplex mcDNA fragments of increasing lengths show that at least 75% of the DNA is organized in an interspersed pattern. In this pattern, single copy sequences with an average length of 800–900 nucleotides are interspersed with repetitive sequences. S1 nuclease digestion of reassociated 3100 nucleotide fragments indicates that 44% of the mcDNA is repetitive and that one-third of the repetitive sequences (average length=285 nucleotides) are interspersed with single copy sequences. We conclude that repetitive sequencies are interspersed with most of the single copy sequences in an interspersion pattern similar to that of Xenopus rahter than to that of another arthropod, Drosophila.Operated by Union Carbide Corporation for the Energy Research and Development Administration     

Deoxyribonucleic acid sequence organization in the genome of the dinoflagellate Crypthecodinium cohnii

Details of the general DNA sequence organization in the dinoflagellate Crypthecodinium cohnii have been obtained by using hydroxylapatite binding experiments, S1 nuclease digestion .and electron microscopy of reassociated DNA. It has been found that roughly half of the genome is made up of unique sequences interspersed with repeated sequence elements with a period of approximately 600 nucleotides. This class represents roughly 95% of the total number of interspersed unique elements in the genome. The remaining 5% are uninterrupted by repeated sequences for at least 4000 nucleotide pairs. The interspersed repeated elements are narrowly distributed in length with 80% under 300 nucleotide pairs in length. About half of the repeated DNA (20-30% of the genome) is not interspersed among unique sequences. The close spacing of the short repeats interspersed throughout much of the genome is consistent with the occurrence of the huge network structures observed in the electron microscope for low Cot reassociation of moderately long fragments. An unusual class of heteroduplexes was detected in the electron microscope which is believed to derive from the reassociation of repeated sequences from different families which are frequently found adjacent to one another in different locations in the genome. The occurrence of this novel arrangement of repeated sequences may reflect the unusual organization of the dinoflagellate nucleus. However, in most respects the sequence arrangement in this unicellular alga is very typical of higher plants and animals.     

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.     

Sequence organization of the human genome

The organization of three sequence classes—single copy, repetitive, and inverted repeated sequences—within the human genome has been studied by renaturation techniques, hydroxylapatite binding methods, and DNA hyperchromism. Repetitive sequence classes are distributed throughout 80% or more of the genome. Slightly more than half of the genome consists of short single copy sequences, with a length of about 2 kb interspersed with repetitive sequences. The average length of the repetitive sequences is also small and approximates the length of these sequences found in other organisms. The sequence organization of the human genome therefore resembles the sequence organization found in Xenopus and sea urchin. The inverted repeats are essentially randomly positioned with respect to both sequence class and sequence arrangement, so that all three sequence classes are found to be mutually interspersed in a portion of the genome.