Mer22-related sequence elements form pericentric repetitive DNA families in primates

We describe a novel repetitive DNA element isolated from three primate species belonging to the family Cercopithecidae. The unusually long 2.6-kb repeat unit of this DNA element is present in high copy number in the pericentromeric region of one pair of chromosomes in both baboon and macaque, forming chromosome-specific satellite-like DNA families. Besides these two very closely related species, the novel DNA element was also detected in the more distantly related African green monkey. However, the copy number of the repeat unit in this species is significantly lower than in macaque and baboon. Sequence analysis revealed that the repeat units of the new repetitive element show similarity to the human MER22 repeat and the Y chromosome-specific TTY2 element, which also exhibits retroelement-like features. Database searches indicate that tandemly arranged MER22-related DNA sequences can also be found in human, raising the possibility that these DNA elements may correspond to a novel primate-specific repetitive DNA group. Recent studies indicate that chromosome-specific pericentric repetitive elements, besides their potential involvement in centromere function, also facilitate homolog recognition during meiosis. In addition, rapid expansion of retroelements in the pericentric regions of chromosomes during interspecific hybridization has been described. In light of these data, we hypothesize that the novel repetitive element described here might have been involved in the speciation of the family Cercopithecidae.     

Medium reiteration frequency repetitive sequences in the human genome.

Fourteen novel medium reiteration frequency (MER) families were found, in the human genome, by using two different methods. Repetition frequencies per haploid human genome were estimated for each of these families as well as for six previously described MER DNA families. By these measurements, the families were found to contain variable numbers of elements, ranging from 200 to 10,000 copies per haploid human genome.     

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3–1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.     

Genomic organization of two families of highly repeated nuclear DNA sequences of maize selected for autonomous replicating activity in yeast

Maize nuclear DNA sequences capable of promoting the autonomous replication of plasmids in yeast were isolated by ligating Eco RI-digested fragments into yeast vectors unable to replicate autonomously. Three such autonomously replicating sequences (ARS), representing two families of highly repeated sequences within the maize genome, were isolated and characterized. Each repetitive family shows hybridization patterns on a Southern blot characteristic of a dispersed sequence. Unlike most repetitive sequences in maize, both ARS families have a constant copy number and characteristic genomic hybridization pattern in the inbred lines examined. Larger genome clones with sequence homology to the ARS-containing elements were selected from a lambda library of maize genomic DNA. There was typically only one copy of an ARS-homologous sequence on each 12–15 kb genomic fragment.     

Repetitive sequences and their organization on genomic clones of Zea mays

Fourteen recombinant clones from Zea mays were studied with regard to their composition of unique and repetitive sequences. Southern hybridization experiments were used to classify restriction fragments of the clones into a unique, middle or highly repetitive class of reiteration frequency. All three classes were often found on the same genomic clone. Crosshybridization studies between clones showed that a given repeat might be present on several clones, and thus four families of highly repetitive elements were established. Heteroduplex analysis was used to show the arrangement and size of repeats common between several clones. A short interspersion pattern of unique, middle and highly repetitive DNA was found. The dispersed repetitive elements were 300-1300 bp in length. Analysis of the pattern produced by a given repeat in genomic Southern experiments suggests that some small dispersed repeats may also exist as part of a larger repeating unit elsewhere in the genome.     

MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis.

The yeast MER1 gene is required for the production of viable meiotic products and for meiotic recombination. Cytological analysis of chromosome spreads from a mer1 mutant indicates that the MER1 gene product is also required for normal chromosome pairing. mer1 strains make axial elements, precursors to the synaptonemal complex; however, the chromosomes in most nuclei do not become fully synapsed. The DNA sequence of the MER1 coding region was determined; the MER1 open reading frame encodes a 270-amino-acid protein with a molecular mass of 31.1 kilodaltons. The MER1 protein shows limited sequence similarity to calmodulin. Expression of the MER1 gene was examined by RNA blot hybridization analysis and through the construction and analysis of mer1::lacZ fusion genes. Expression of the MER1 gene is meiotically induced and required the IME1 gene product. Thus, expression of the MER1 gene early in meiosis is required for proper chromosome pairing and meiotic recombination.     

Tissue-specific and age-related variations in repetitive sequences of mouse extrachromosomal circular DNAs

Extrachromosomal circular (ecc) DNA was isolated from mouse brain, liver, and heart tissues at different ages. To determine the abundance of repetitive sequences in eccDNAs, preparations were probed for short-interspersed (B1 and B2), long-interspersed (L1), endogenous retroviral-like (IAP), and tandemly repeated satellite sequences (SAT) of the mouse genome. Together these sequence families comprise approximately 15% of the mouse genome. The hybridization results showed that each tissue had a characteristic pattern of repetitive sequence elements in eccDNAs, and the abundance of repetitive sequences changed as a function of age. Repetitive sequences decreased in liver and brain eccDNAs from 1 month to 8 months of age but appeared to remain stable thereafter. In contrast, repetitive sequence families in heart eccDNAs were constant from 1 month to 16 months of age but declined in 24-month-old mice. The present studies indicate that extrachromosomal sequences exhibit greater flexibility than chromosomal sequences.     

Analysis of transposons and repeat composition of the sunflower (Helianthus annuus L.) genome

A sample-sequencing strategy combined with slot–blot hybridization and FISH was used to study the composition of the repetitive component of the sunflower genome. One thousand six hundred thirty-eight sequences for a total of 954,517 bp were analyzed. The fraction of sequences that can be classified as repetitive using computational and hybridization approaches amounts to 62% in total. Almost two thirds remain as yet uncharacterized in nature. Of those characterized, most belong to the gypsy superfamily of LTR-retrotransposons. Unlike in other species, where single families can account for large fractions of the genome, it appears that no transposon family has been amplified to very high levels in sunflower. All other known classes of transposable elements were also found. One family of unknown nature (contig 61) was the most repeated in the sunflower genome. The evolution of the repetitive component in the Helianthus genus and in other Asteraceae was studied by comparative analysis of the hybridization of total genomic DNAs from these species to the sunflower small-insert library and compared to gene-based phylogeny. Very little similarity is observed between Helianthus species and two related Asteraceae species outside of the genus. Most repetitive elements are similar in annual and perennial Helianthus species indicating that sequence amplification largely predates such divergence. Gypsy-like elements are more represented in the annuals than in the perennials, while copia-like elements are similarly represented, attesting a different amplification history of the two superfamilies of LTR-retrotransposons in the Helianthus genus.     

Isolation and sequence analysis ofCaenorhabditis briggsae repetitive elements related to theCaenorhabditis elegans transposon Tc1

Summary We have identified two repetitive element families in the genome of the nematodeCaenorhabditis briggsae with extensive sequence identity to theCaenorhabditis elegans transposable element Tc1. Five members each of the TCb1 (previously known as Barney) and TCb2 families were isolated by hybridization to a Tc1 probe. Tc1-hybridizing repetitive elements were grouped into either the TCb1 or TCb2 family based on cross-hybridization intensities among theC. briggsae elements. The genomic copy number of the TCb1 family is 15 and the TCb2 family copy number is 33 in theC. briggsae strain G16. The two transposable element families show numerous genomic hybridization pattern differences between twoC. briggsae strains, suggestive of transpositional activity. Two members of the TCb1 family, TCb1#5 and TCb1#10, were sequenced. Each of these two elements had suffered an independent single large deletion. TCb1#5 had a 627-bp internal deletion and TCb1#10 had lost 316 bp of one end. The two sequenced TCb1 elements were highly conserved over the sequences they shared. A 1616-bp composite TCb1 element was constructed from TCb1#5 and TCb1#10. The composite TCb1 element has 80-bp terminal inverted repeats with three nucleotide mismatches and two open reading frames (ORFs) on opposite strands. TCb1 and the 1610-bp Tc1 share 58% overall nucleotide sequence identity, and the greatest similarity occurs in their ORF1 and inverted repeat termini.     

Highly repetitive DNA sequences in cyanobacterial genomes.

We characterized three distinct families of repeated sequences in the genome of the cyanobacterium Calothrix sp. strain PCC 7601. These repeated sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides. These elements were named short tandemly repeated repetitive (STRR) sequences. We used the three different Calothrix STRR sequences as probes to perform Southern hybridization experiments with DNAs extracted from various cyanobacterial strains, Bacillus subtilis, and Escherichia coli. The three different STRR sequences were found as repetitive genomic DNA components specific to the heterocystous strains tested. The role of the STRR sequences, as well as their possible use in taxonomic studies, is discussed.     

Nucleotide sequence of a repetitive element isolated from Leptospira interrogans serovar hardjo type hardjo-bovis.

A repetitive element from the genome of Leptospira interrogans serovar hardjo type hardjo-bovis ('L. hardjo-bovis') was identified, cloned and sequenced. Similar sequences were shown by hybridization to be encoded by a further eight of 32 other leptospiral serovars tested. An undefined number of repetitive elements were located in the L. hardjo-bovis genome; sequence degeneracy of the elements was observed and no significant open reading frames were identified within the AT-rich (60%) 1467 bp repetitive element. The termini encoded a GC-rich 8 bp repeat motif and two variants showed rearrangements centred on these motifs. The nucleotide sequences of the chromosomal regions flanking the repetitive elements were determined but showed no similarities, with one exception which had a GAAC repeat directly adjacent to both termini. Similar hybridization patterns were shown by Southern transfers of L. hardjo-bovis total genomic digests probed with the repetitive element. Oligonucleotide primer pairs designed from sequences internal to the repetitive element and adjacent chromosomal regions were used in polymerase chain reaction experiments. With one primer pair all L. hardjo-bovis isolates, but no other serovar, gave identical amplified products. Evidence that the repetitive element may have derived from an acquired insertion sequence that is now inactive and chromosomally fixed is discussed.     

Characterisation of a short interspersed repeat (Mermaid) that has family members on human chromosome 21 and elsewhere in the human genome

To understand the architecture of the human genome, we need a complete definition of all the repeat sequence families, as these make up the majority of human DNA. We have isolated a small DNA fragment from human chromosome 21 and have used sequence analysis of this fragment to uncover a new low copy repeat element of approximately 300 bp that we term the Mermaid repeat. This repeat is related to, but is different from, the MER 12 repeat and is interspersed in the genome. Mermaid family members that we have studied are between 81%–87% identical to our preliminary consensus sequence. Therefore, we have added a new member to the large collection of human repetitive elements. In addition, we have mapped a Mermaid repeat to a telomeric position on the long arm of human chromosome 21, at 21q22.3     

Highly repetitive DNA families restricted to germ cells in a Japanese hagfish (Eptatretus burgeri): a hierarchical and mosaic structure in eliminated chromosomes

It is known that in eight hagfishes chromosome elimination occurs during early embryogenesis. The eliminated chromosomes are mostly C-band positive, so that none of the somatic cells have any C-band-positive chromatin. Recently, some highly repetitive DNA sequences have been reported as eliminated elements in these hagfishes based on molecular biological methods. However, no germline-restricted repetitive DNA have been directly isolated from the Japanese hagfish Eptatretus burgeri, from which approximately 21% of the total DNA is eliminated from presumptive somatic cells. Through electrophoretic investigation after digestion with restriction endonucleases, two DNA families that are restricted to germline DNA were isolated. Molecular cloning and sequence analysis revealed that these families are composed of closely related sequences of 64 and 57bp in length, respectively. Southern blot hybridization revealed that the two DNA families are restricted to germline DNA and were thus named EEEb1 and EEEb2, respectively. Moreover, these eliminated elements were highly and tandemly repeated, and it is predicted that they might amplify by saltatory replication and have evolved in a concerted manner. By densitometric scanning, EEEb1 and EEEb2 were found to amount to make up approximately 18.5 and 0.024% of the total germline genomic DNA, accounting for 88.6% of the total eliminated DNA. A fluorescence in situ hybridization experiment demonstrated that EEEb1 is located on all C-band-positive chromosomes that are limited to germ cells, suggesting that EEEb1 is the primary component of eliminated DNA of E. burgeri.     

Diverse patterns of the tandem repeats organization in rye chromosomes

Although the monomer size, nucleotide sequence, abundance and species distribution of tandemly organized DNA families are well characterized, little is known about the internal structure of tandem arrays, including total arrays size and the pattern of monomers distribution. Using our rye specific probes, pSc200 and pSc250, we addressed these issues for telomere associated rye heterochromatin where these families are very abundant. Fluorescence in situ hybridization (FISH) on meiotic chromosomes revealed a specific mosaic arrangement of domains for each chromosome arm where either pSc200 or pSc250 predominates without any obvious tendency in order and size of domains. DNA of rye-wheat monosomic additions studied by pulse field gel electrophoresis produced a unique overall blot hybridization display for each of the rye chromosomes. The FISH signals on DNA fibres showed multiple monomer arrangement patterns of both repetitive families as well as of the Arabidopsis-type telomere repeat. The majority of the arrays consisted of the monomers of both families in different patterns separated by spacers. The primary structure of some spacer sequences revealed scrambled regions of similarity to various known repetitive elements. This level of complexity in the long-range organization of tandem arrays has not been previously reported for any plant species. The various patterns of internal structure of the tandem arrays are likely to have resulted from evolutionary interplay, array homogenization and the generation of heterogeneity mediated by double-strand breaks and associated repair mechanisms.     

New families of site-specific repetitive DNA sequences that comprise constitutive heterochromatin of the Syrian hamster (Mesocricetus auratus, Cricetinae, Rodentia)

We molecularly cloned new families of site-specific repetitive DNA sequences from BglII- and EcoRI-digested genomic DNA of the Syrian hamster (Mesocricetus auratus, Cricetrinae, Rodentia) and characterized them by chromosome in situ hybridization and filter hybridization. They were classified into six different types of repetitive DNA sequence families according to chromosomal distribution and genome organization. The hybridization patterns of the sequences were consistent with the distribution of C-positive bands and/or Hoechst-stained heterochromatin. The centromeric major satellite DNA and sex chromosome-specific and telomeric region-specific repetitive sequences were conserved in the same genus (Mesocricetus) but divergent in different genera. The chromosome-2-specific sequence was conserved in two genera, Mesocricetus and Cricetulus, and a low copy number of repetitive sequences on the heterochromatic chromosome arms were conserved in the subfamily Cricetinae but not in the subfamily Calomyscinae. By contrast, the other type of repetitive sequences on the heterochromatic chromosome arms, which had sequence similarities to a LINE sequence of rodents, was conserved through the three subfamilies, Cricetinae, Calomyscinae and Murinae. The nucleotide divergence of the repetitive sequences of heterochromatin was well correlated with the phylogenetic relationships of the Cricetinae species, and each sequence has been independently amplified and diverged in the same genome.     

Evolutionary change in the repetition frequency of sea urchin DNA sequences

The frequency of occurrence of particular repetitive sequence families has been estimated in the DNA of the three sea urchin species Strongylocentrotus purpuratus, Strongylocentrotus franciscanus and Lytechinus pictus using individual cloned S. purpuratus repetitive sequence elements. Cloned repetitive sequence elements as described by Scheller et al. (1977a) were prepared by reassociation of S. purpuratus DNA fragments to repetitive Cot, digestion with single-strand-specific nuclease S1 and ligation of synthetic restriction sites to their ends. The sequences were cloned by insertion at the Eco RI site of plasmid RSF2124, labeled, strand-separated and reassociated with 800–900 nucleotide long unlabeled DNA. Both kinetic (genomic DNA excess) and saturation (cloned DNA excess) estimates of frequencies were made. For nine cloned fragments, the ratio of the repetition frequency in S. purpuratus DNA to that in S. franciscanus DNA ranges from about 20 to about 1. In the four cases examined, only a few copies were detected in the DNA of L. pictus. Estimates have also been made of frequency changes in many repetitive families by measuring the reassociation of labeled repetitive DNA fractions of each species with total DNA from other species. In each reciprocal comparison, the labeled repetitive sequences reassociate more slowly with DNA of other species than with DNA of the species from which they were prepared. Thus it appears that the dominant repetitive sequence families in the DNA of each species are present at lower frequencies in the DNA of closely related species. Measurements of thermal stability have been made of S. purpuratus cloned repetitive sequences reassociated with S. franciscanus DNA or S. purpuratus DNA. Most families have changed both in frequency and sequence, although some have changed little in sequence but show great changes in frequency.     

Characterization of the primate-specific repetitive DNA element MER1.

Computer analyses of the 3'-flanking DNA sequence of the human elastase I gene revealed a significant degree of similarity with seven human gene sequences in the GenBank and EMBL databases. Genomic Southern analysis indicates that the shared nucleotide sequences are a primate-specific family of short interspersed elements. These elements are members of MER1 sequences (medium reiteration frequency sequences). The consensus sequence of MER1 repeats spans 543 nucleotides and contains several inverted repeats. Since the copy number of MER1 elements seems to be much smaller than that of Alu and L1 repeats, MER1 elements may provide useful landmarks marks for human genome mapping.