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.     

A chromosome 5-specific repetitive DNA sequence in rice (Oryza sativa L)

Repetitive DNA sequences in the rice genome comprise more than half of the nuclear DNA. The isolation and characterization of these repetitive DNA sequences should lead to a better understanding of rice chromosome structure and genome organization. We report here the characterization and chromosome localization of a chromosome 5-specific repetitive DNA sequence. This repetitive DNA sequence was estimated to have at least 900 copies. DNA sequence analysis of three genomic clones which contain the repeat unit indicated that the DNA sequences have two sub-repeat units of 37 bp and 19 bp, connected by 30-to 90-bp short sequences with high similarity. RFLP mapping and physical mapping by fluorescence in situ hybridization (FISH) indicated that almost all copies of the repetitive DNA sequence are located in the centromeric heterochromatic region of the long arm of chromosome 5. The strategy for cloning such repetitive DNA sequences and their uses in rice genome research are discussed.     

A new rice repetitive DNA shows sequence homology to both 5S RNA and tRNA.

Moderately repetitive DNA sequences are found in the genomes of all eucaryotes that have been examined. We now report the discovery of a novel, transcribed, moderately repetitive DNA sequence in a higher plant which is different from any of the known repetitive DNA sequences from any organism. We isolated a rice cDNA clone which hybridizes to multiple bands on genomic blot analysis. The sequence of this 352 bp cDNA contains four regions of homology to the wheat phenylalanine tRNA, including the polymerase III-type promoter. Unexpectedly, two regions of the same 352 bp sequence also show homology to the wheat 5S RNA sequence. Using the cDNA as a probe, we have isolated six genomic clones which contain long tandem repeats of 355 bp sequence, and have sequenced nine repeat units. Our findings suggest that the rice repetitive sequence may be an amplified pseudogene with sequence homology to both 5S RNA and tRNA, but organized as long tandem repeats resembling 5S RNA genes. This is the first example showing homology between the sequences of a moderately repetitive DNA with unknown function and 5S RNA.     

Organization of the micronuclear genome of oxytricha nova

In the hypotrichous ciliated protozoan Oxytricha nova, approximately 95% of the micronuclear genome, including all of the repetitive DNA and most of the unique sequence DNA, is eliminated during the formation of the macronuclear genome. We have examined the interspersion patterns of repetitive and unique and eliminated and retained sequences in the micronuclear genome by characterizing randomly selected clones of micronuclear DNA. Three major classes of clones have been defined: (1) those containing primarily unique, retained sequences; (2) those containing only unique, eliminated sequences; and (3) those containing only repetitive, eliminated sequences. Clones of type one and three document two aspects of organization observed previously: clustering of macronuclear destined sequences and the presence of a prevalent repetitive element. Clones of the second type demonstrate for the first time that eliminated unique sequence DNA occurs in long stretches uninterrupted by repetitive sequences. To further examine repetitive sequence interspersion, we characterized the repetitive sequence family that is present in 50% of the clones (class three above). A consensus map of this element was obtained by mapping approximately 80 phage clones and by hybridization to digests of micronuclear DNA. The repeat element is extremely large (approximately 24 kb) and is interspersed with both macronuclear destined sequences and eliminated unique sequences.     

Repetitive sequences in Eurasian lynx (Lynx lynx L.) mitochondrial DNA control region

Mitochondrial DNA (mtDNA) control region (CR) of numerous species is known to include up to five different repetitive sequences (RS1-RS5) that are found at various locations, involving motifs of different length and extensive length heteroplasmy. Two repetitive sequences (RS2 and RS3) on opposite sides of mtDNA central conserved region have been described in domestic cat (Felis catus) and some other felid species. However, the presence of repetitive sequence RS3 has not been detected in Eurasian lynx (Lynx lynx) yet. We analyzed mtDNA CR of 35 Eurasian lynx (L. lynx L.) samples to characterize repetitive sequences and to compare them with those found in other felid species. We confirmed the presence of 80 base pairs (bp) repetitive sequence (RS2) at the 5' end of the Eurasian lynx mtDNA CR L strand and for the first time we described RS3 repetitive sequence at its 3' end, consisting of an array of tandem repeats five to ten bp long. We found that felid species share similar RS3 repetitive pattern and fundamental repeat motif TACAC.     

Repetitive sequence environment distinguishes housekeeping genes

Housekeeping genes are expressed across a wide variety of tissues. Since repetitive sequences have been reported to influence the expression of individual genes, we employed a novel approach to determine whether housekeeping genes can be distinguished from tissue-specific genes by their repetitive sequence context. We show that Alu elements are more highly concentrated around housekeeping genes while various longer (> 400-bp) repetitive sequences (“repeats”), including Long Interspersed Nuclear Element-1 (LINE-1) elements, are excluded from these regions. We further show that isochore membership does not distinguish housekeeping genes from tissue-specific genes and that repetitive sequence environment distinguishes housekeeping genes from tissue-specific genes in every isochore. The distinct repetitive sequence environment, in combination with other previously published sequence properties of housekeeping genes, was used to develop a method of predicting housekeeping genes on the basis of DNA sequence alone. Using expression across tissue types as a measure of success, we demonstrate that repetitive sequence environment is by far the most important sequence feature identified to date for distinguishing housekeeping genes.     

A family of Saccharomyces cerevisiae repetitive autonomously replicating sequences that have very similar genomic environments

We have characterized a family of moderately repetitive autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Restriction mapping, deletion studies and hybridization studies suggest that these ARSs, which are probably less than 350 base-pairs in size, share one common feature: each is located close to, but not within, a repetitive sequence (131) of approximately 10(3) to approximately 1.5 X 10(3) base-pairs in length. These ARSs can be divided into two classes (X and Y) by their sequence homology and genomic environments. Each of the class X ARSs is embedded within a repetitive sequence (X) of variable length (approximately 0.3 X 10(3) to approximately 3.75 X 10(3) base-pairs); each of the class Y ARSs is embedded within a highly conserved repetitive sequence (Y) of approximately 5.2 X 10(3) base-pairs in length. Both of these sequences are located directly adjacent to the 131 sequence.     

Sex-specific organisation of middle repetitive DNA sequences in the mealybug Planococcus lilacinus.

Differential organisation of homologous chromosomes is related to both sex determination and genomic imprinting in coccid insects, the mealybugs. We report here the identification of two middle repetitive sequences that are differentially organised between the two sexes and also within the same diploid nucleus. These two sequences form a part of the male-specific nuclease-resistant chromatin (NRC) fraction of a mealybug Planococcus lilacinus. To understand the phenomenon of differential organisation we have analysed the components of NRC by cloning the DNA sequences present, deciphering their primary sequence, nucleosomal organisation, genomic distri-bution and cytological localisation. Our observations suggest that the middle repetitive sequences within NRC are functionally significant and we discuss their probable involvement in male-specific chromatin organisation.     

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.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Essential role of duplications of short motif sequences in the genomic evolution of Bombyx mori

Summary The Bombyx fibroin gene has a discrete mosaic structure of various repetitive sequences, which may have evolved through various repeating arrangements. Detailed sequence analysis of the fibroin gene containing coding and noncoding regions revealed that the whole sequence could be arranged as an array of short repetitive sequences. A portion of the intron of the fibroin gene is one of interspersed repetitive elements. We cloned a 1.5-kb DNA fragment of the Bombyx genome that contains interspersed elements homologous to the intron sequence. Sequence comparison between the intron and the 1.5-kb fragment shows that partial duplication has frequently occurred in evolutionary progress, and the resultant repetitive blocks of short motif sequences are abundant in the genome. These facts suggest that tandem duplication of the short motif sequence is an important rearrangement in genomic evolution of the fibroin gene. Offprint requests to: S. Ichimura     

Modeling repetitive,non‐globular proteins

While ab initio modeling of protein structures is not routine, certain types of proteins are more straightforward to model than others. Proteins with short repetitive sequences typically exhibit repetitive structures. These repetitive sequences can be more amenable to modeling if some information is known about the predominant secondary structure or other key features of the protein sequence. We have successfully built models of a number of repetitive structures with novel folds using knowledge of the consensus sequence within the sequence repeat and an understanding of the likely secondary structures that these may adopt. Our methods for achieving this success are reviewed here.     

A comprehensive examination of protein sequences for evidence of internal gene duplication

Summary We have implemented a routine procedure for screening protein sequences for evidence of intragenic duplications. We tested 163 protein sequences representing 116 superfamilies of unrelated proteins. Twenty superfamilies contain proteins with internal gene duplications. The intragenic duplications detected can be divided into two major types. (1) One or more duplications of all or part of a gene produce a protein with two or several detectable regions of sequence homology. Sequences from 18 superfamilies contained this type of duplication. (2) Repeated reduplication of a small DNA segment can produce a protein that is repetitive over most of its length. Three superfamilies contain such repetitive sequences. We also investigated the limits of detection of ancient duplications using sequences derived by random mutation of a model sequence consisting of ten 10-residue repeats. The original repetitive nature of the sequence was usually detected after 250 point mutations even though the ancestral segment could not be accurately reconstructed.     

CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.     

A whole-genome snapshot of 454 sequences exposes the composition of the barley genome and provides evidence for parallel evolution of genome size in wheat and barley

The genomes of barley and wheat, two of the world's most important crops, are very large and complex due to their high content of repetitive DNA. In order to obtain a whole-genome sequence sample, we performed two runs of 454 (GS20) sequencing on genomic DNA of barley cv. Morex, which yielded approximately 1% of a haploid genome equivalent. Almost 60% of the sequences comprised known transposable element (TE) families, and another 9% represented novel repetitive sequences. We also discovered high amounts of low-complexity DNA and non-genic low-copy DNA. We identified almost 2300 protein coding gene sequences and more than 660 putative conserved non-coding sequences. Comparison of the 454 reads with previously published genomic sequences suggested that TE families are distributed unequally along chromosomes. This was confirmed by in situ hybridizations of selected TEs. A comparison of these data for the barley genome with a large sample of publicly available wheat sequences showed that several TE families that are highly abundant in wheat are absent from the barley genome. This finding implies that the TE composition of their genomes differs dramatically, despite their very similar genome size and their close phylogenetic relationship.     

Human transposon-like elements insert at a preferred target site: evidence for a retrovirally mediated process.

Members of the human transposon-like family of repetitive sequences (called THE 1 repeats) like many other repetitive DNA sequences are flanked by short direct repeats. Comparison of the base sequences of twelve examples of these flanking direct repeats indicates that THE 1 repeats insert into a preferred genomic target site. In one case, we have identified the sequence of an empty site into which a THE 1 element inserted. The sequence of this empty site and sequences of truncated THE 1 LTRs are consistent with a retroviral mechanism for the insertion of THE 1 elements. Truncated transposon structures illustrate for the first time that intermediate structures of retrotransposition may also be integrated into the genome.     

CetSINEs and AREs are not SINEs but are parts of cetartiodactyl L1

Short interspersed repetitive elements (SINEs) are widely distributed among the genomes of eukaryotes. We proposed previously that a SINE should be defined by the presence of a region homologous to a tRNA or to 7SL RNA, together with A-box and B-box promoter sequences, in order to distinguish SINEs from other short repetitive sequences, such as short segments of LINEs (long interspersed repetitive elements; Okada et al. Gene 205, 229–243, 1997). Numerous SINE sequences have been deposited to date in DNA databases. In some cases, however, designation of a particular sequence is problematic when the short repetitive sequence has been defined as a SINE without reference to the presence or absence of promoter elements specific for RNA polymerase III. We demonstrate here that four different sequences, namely, ARE1p, ARE2p, CetSINE1, and CetSINE2, each of which has been reported as a SINE, are, in fact, only partial sequences of members of a new subfamily of L1. We also demonstrate that members of this subfamily are distributed specifically among the genomes of cetartiodactyls. Received: 3 May 2000 / Accepted: 22 August 2000