Dispersed repetitive sequences of the mouse genome are differentially represented in extrachromosomal circular DNAs in vivo

Eukaryotic cells contain extrachromosomal circular (ecc) DNAs which are homologous to chromosomal sequences. We have isolated eccDNAs from whole tissues of C57BL/6 mice. Using hybridization techniques, we show that R-, MIF-, B1-, and B2-dispersed repetitive sequences of the mouse genome are differentially represented in heart, brain, and liver tissues. Moreover, we show that the relative abundance of B2 sequences in heart and liver eccDNAs is higher than the relative abundance of the other repetitive sequence families studied.     

A cluster of repetitive elements within a 700 base pair region in the mouse genome

Approximately 39% of the clones from a BALB/c mouse genomic library hybridized with polyadenylated cytoplasmic RNA extracted from anemic mouse spleen. The DNA sequence of a portion of one such clone revealed the presence of three repetitive sequence elements within a 700 bp span. All three elements contain putative RNA polymerase III control regions oriented in the same direction and oligo(dA) tracts at their 3' ends. The first element is a member of the murine B1 family. A comparison of this element with other B1 family members indicates that the B1 family can be divided into two subclasses based on commonly held base changes and deletions. The second element within this 700 bp region may be a member of a new murine Alu family. Its structure is analogous to other murine Alu-equivalent sequences with respect to overall length, the presence of a 3' oligo(dA) tract and putative RNA polymerase III control regions. The third element is a murine type 2 Alu-equivalent sequence.     

Palindromic repetitive elements in the mitochondrial genome of Volvox

Group I introns were found in the cob and cox I genes of Volvox carteri. These introns contain tandem arrays of short palindromic sequences that are related to each other. Inspection of other regions in the mtDNA revealed that similar palindromic repetitive sequences are dispersed in the non-protein coding regions of the mitochondrial genome. Analysis of the group I intron in the cob gene of another member of Volvocaceae, Volvox aureus, has shown that its sequence is highly homologous to its counterpart in V. carteri with the exception of a cluster of palindromic sequences not found in V. carteri. This indicates that the palindromic clusters were inserted into the introns after divergence of the two species, presumably due to frequent insertions of the palindromic elements during evolution of the Volvocaceae. Possible involvement of the palindromic repetitive elements in the molecular evolution of functional RNAs is discussed.     

Microsatellites and associated repetitive elements in the sheep genome

To determine the frequency and clustering of a variety of simple di-and trinucleotide repeats, an Artiodactyl short interspersed element (SINE), an ovine satellite repeat, and a human Alu 1 repeat were used to screen a random selection of cosmids containing inserts of ovine genomic DNA. In total, 197 individual cosmids were digested with EcoRI and the fragments separated on 0.7% agarose gels. Southern blots of these gels were then sequentially probed with (AC)₇, (CT)₉, and (CAC)₆ oligonucleotides, and the repeats described above. The frequency at which (AC)₁, (CT)_n, and (CAC)_n repeats were found in the cosmids indicated that they occurred at average intervals of 65 kb, 367 kb, and 213 kb respectively within the ovine genome. The Artiodactyl SINE was the most common, occurring at an average interval of 20 kb. No human Alu 1 sequences were detected. There was a significant positive association between the (AC)_n and the Artiodactyl SINE. This association is quite strong as there was significant clustering of the two repeats both within cosmids and also within the EcoRI fragments of the digested genomic fragments. With the exception of the sheep satellite sequence, which occurs in tandem arrays, none of the other repeats showed significant clustering within the 41-kb (average size) cosmid inserts. The first 25 ovine microsatellites we characterized had an average polymorphic information content (PIC) of 0.65. The different microsatellite types, containing either perfect, imperfect, or compound repeats, had similar average PICs of 0.64, 0.65, and 0.66 respectively. There was a weak regression relationship (R²(adj)%=21.9) between the length of the longest uninterrupted dinucleotide repeat in the largest allele and the PIC of the microsatellite.     

Duplicated region of the mouse genome containing a cytoplasmic gamma-actin processed pseudogene associated with long interspersed repetitive elements

The structures of two cloned recombinants of bacteriophage lambda and mouse genomic DNA (lambda mA14 and lambda mA36) were compared by electron microscopic analysis of various heteroduplex DNAs, restriction endonuclease mapping and nucleotide sequence determination. Each clone was shown to be derived from a distinct region of the mouse genome, but the two exhibited structural similarity over a region of at least 11,000 bases which included a cytoskeletal gamma-actin processed pseudogene of approximately 1800 bases. It is concluded that the two genomic regions were derived from a common ancestral region by duplication or amplification. The homologous regions of the two clones contained members of the long interspersed repetitive L1Md (long interspersed repeated sequence 1 of Mus domesticus) family lying in opposite orientation to one another, so that single-stranded DNA from the clones could form intra-molecular heteroduplexes. The complete nucleotide sequences of three L1Md members in lambda mA14 were determined. The longest of these (L1Md-14LH) had inserted into the gamma-actin processed pseudogene and, although it contained internal deletions, appeared to possess intact 5' and 3' ends. A second L1Md member (L1Md-14RH1) also appeared to have an intact 5' end but had lost most of its 3' portion, and a third member (L1Md-14RH2) was an internal fragment. The repeated sequence at the 5' ends of L1Md-14LH and L1Md-14RH1 showed these to be members of the L1Md-A family.     

Novel families of interspersed repetitive elements from the human genome.

Six novel families of interspersed repetitive elements have been detected in the available human DNA sequences using computer-assisted analyses. The estimated total number of elements in the reported six families is over 17,000. Sequences representative for each family range from approximately 150 to 650 base pairs (bp) in length and are predominantly (A + T)-rich. Sequences from four families contain stretches of patchy complementarity up to 45 bp long. Member of one of the families is likely be directly involved in a multigene deletion on chromosome 14. Two of the six sequence families are homologous to 'low reiteration frequency sequences' from monkey cells, detected first in defective variants of simian virus 40. Like Alu and L1 families, the newly discovered families are probably composed of pseudogenes derived from functional genes.     

Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk- DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.     

Integration of a vector containing rodent repetitive elements in the rat genome.

We have previously shown that integration of a polyoma vector containing rodent repetitive elements into rat cellular DNA is non-random (Wallenburg et al. J. Virol. 50: 678-683). Junctions between the polyoma vector and the host DNA occur in the repetitive sequences of the vector about ten times more frequently than would be expected if sequences from the vector were used randomly for integration. In this paper we looked at the host sequences involved in these junctions. Our analysis did not reveal any repetitive or specific sequences and we presume therefore that the repetitive sequences of the vector acted as hot spots for illegitimate recombination. We also analysed the integration mechanism and found that: First, even though the polyoma vector was transfected in the presence of carrier DNA, integration did not involve the formation of a transgenome. Second, in at least one of the clones analysed, integration resulted in deletion of host DNA sequences. Third, the host DNA displaced at the integration site was considerably longer than the integrated segment.     

A new family of interspersed repetitive DNA sequences in the mouse genome

Repetitive DNA sequences near immunoglobulin genes in the mouse genome (Steinmetz et al., 1980a,b) were characterized by restriction mapping and hybridization. Six sequences were determined that turned out to belong to a new family of dispersed repetitive DNA. From the sequences, which are called R1 to R6, a 475 base-pair consensus sequence was derived. The R family is clearly distinct from the mouse B1 family (Krayev et al., 1980). According to saturation hybridization experiments, there are about 100,000 R sequences per haploid genome, and they are probably distributed throughout the genome. The individual R sequences have an average divergence from the consensus sequence of 12.5%, which is largely due to point mutations and, among those, to transitions. Some R sequences are severly truncated. The R sequences extend into A-rich sequences and are flanked by short direct repeats. Also, two large insertions in the R2 sequence are flanked by direct repeats. In the neighbourhood of and within R sequences, stretches of DNA have been identified that are homologous to parts of small nuclear RNA sequences. Mouse satellite DNA-like sequences and members of the B1 family were also found in close proximity to the R sequences. The dispersion of R sequences within the mouse genome may be a consequence of transposition events. The possible role of the R sequences in recombination and/or gene conversion processes is discussed.     

Methylation dynamics of repetitive DNA elements in the mouse germ cell lineage

Repetitive DNA elements account for a substantial fraction of the mammalian genome. Many are subject to DNA methylation, which is known to undergo dynamic change during mouse germ cell development. We found that repeat sequences of three different classes retain high levels of methylation at E12.5, when methylation is erased from many single-copy genes. Maximal demethylation of repeats was seen later in development and at different times in male and female germ cells. At none of the time points examined (E12.5, E15.5, and E17.5) did we see complete demethylation, suggesting that methylation patterns on repeats may be passed on from one generation to the next. In male germ cells, we observed a de novo methylation event resulting in complete methylation of all the repeats in the interval between E15.5 and E17.5, which was not seen in females. These results suggest that repeat sequences undergo coordinate changes in methylation during germ cell development and give further insights into germ cell reprogramming in mice.     

Unique profile of ordered arrangements of repetitive elements in the C57BL/6J mouse genome implicating their functional roles

The entirety of all protein coding sequences is reported to represent a small fraction (~2%) of the mouse and human genomes; the vast majority of the rest of the genome is presumed to be repetitive elements (REs). In this study, the C57BL/6J mouse reference genome was subjected to an unbiased RE mining to establish a whole-genome profile of RE occurrence and arrangement. The C57BL/6J mouse genome was fragmented into an initial set of 5,321 units of 0.5 Mb, and surveyed for REs using unbiased self-alignment and dot-matrix protocols. The survey revealed that individual chromosomes had unique profiles of RE arrangement structures, named RE arrays. The RE populations in certain genomic regions were arranged into various forms of complexly organized structures using combinations of direct and/or inverse repeats. Some of these RE arrays spanned stretches of over 2 Mb, which may contribute to the structural configuration of the respective genomic regions. There were substantial differences in RE density among the 21 chromosomes, with chromosome Y being the most densely populated. In addition, the RE array population in the mouse chromosomes X and Y was substantially different from those of the reference human chromosomes. Conversion of the dot-matrix data pertaining to a tandem 13-repeat structure within the Ch7.032 genome unit into a line map of known REs revealed a repeat unit of ~11.3 Kb as a mosaic of six different RE types. The data obtained from this study allowed for a comprehensive RE profiling, including the establishment of a library of RE arrays, of the reference mouse genome. Some of these RE arrays may participate in a spectrum of normal and disease biology that are specific for mice.     

Structural organization of the tissue-specific middle repetitive sequence of the mouse genome

The structural genes closely linked to the particular middle repetitive sequence (MRS) expressed in liver nuclei were cloned from the mouse genomic library. From one-fourth of 3,200 MRS-containing clones, 21 clones were obtained as mRNA coding sequence-linked MRS clones. From examination of the structural organization and specificity of expression of the MRS and the mRNA coding sequence, it was concluded that expressions of the MRS and the structural genes closely linked to the MRS are independently regulated.     

Chromosomal rearrangements associated with LINE elements in the mouse genome.

Two segments of DNA that have apparently inserted in the interval between the two adult beta-globin genes in BALB/c (Hbbd haplotype) but not in C57B1/10 (Hbbs haplotype) mouse strains have been described (1). These putative insertions, each about 1000 bp in length, mapped near a repetitive element. To determine the precise position of these alleged insertions, their target sites, and the nature of their boundaries, we cloned and sequenced the appropriate regions of both chromosomes. One of the two segments is not an insertion but rather a region between two independently integrated L1 repetitive elements (LINEs) (2), one in Hbbd and the other in the Hbbs chromosome. The other segment is an insertion of 940 bp which is located within the L1 element in the Hbbd chromosome. This insert is unusual in that it exists in only one copy in the BALB/c genome.     

Dispersed repetitive DNA sequence of Mucor racemosus.

A dispersed repetitive DNA sequence has been identified within the genome of the fungus Mucor racemosus. Recombinant phage clones, as well as a plasmid harboring the sequence, have been isolated. Examination of cloned fragments comprising part of the repetitive sequence has led to a partial characterization of the element. The sequence has been detected in other Mucor species, and although the apparent number and chromosomal position of the repetitive sequence vary from strain to strain, it is clear that at least portions of the element have been conserved.     

P53 and the defenses against genome instability caused by transposons and repetitive elements

The recent publication by Wylie et al. is reviewed, demonstrating that the p53 protein regulates the movement of transposons. While this work presents genetic evidence for a piRNA‐mediated p53 interaction with transposons in Drosophila and zebrafish, it is herein placed in the context of a decade or so of additional work that demonstrated a role for p53 in regulating transposons and other repetitive elements. The line of thought in those studies began with the observation that transposons damage DNA and p53 regulates DNA damage. The presence of transposon movement can increase the rate of evolution in the germ line and alter genes involved in signal transduction pathways. Transposition can also play an important role in cancers where the p53 gene function is often mutated. This is particularly interesting as recent work has shown that de‐repression of repetitive elements in cancer has important consequences for the immune system and tumor microenvironment.     

Evolution of a mouse Y chromosomal sequence flanked by highly repetitive elements

Mammalian primary sex is determined by the presence or absence of the Y chromosome. However, little is known about the molecular processes through which the Y chromosome exerts its action. We applied recombinant DNA techniques to isolate mouse Y chromosomal fragments and described previously a clone designated as AC11 (Y. Nishioka and E. Lamothe. 1986. Genetics, 113:417-432). To obtain information on DNA sequences that flank AC11, we screened a mouse genomic library for the presence of AC11-related sequences and isolated over 50 positive clones. In this report we describe clones ACC2 and ACC3, both of which contain highly repetitive elements. Using a male-specific portion of these clones, we compared DNA's isolated from mice (Mus musculus, M. hortulanus, M. spretus, M. cookii, M. pahari, and M. platythrix), rat, hamster, and guinea pig and obtained results that agree with the phylogenetic relationships deduced from morphological and biochemical studies. The male-specific accumulation of the related sequences was found only in M. musculus, M. hortulanus, and M. spretus.     

Genetic exchange between endogenous and exogenous LINE-1 repetitive elements in mouse cells.

The repetitive LINE (L1) elements of the mouse, which are present at about 10(5) copies per genome and share over 80% of sequence homology, were examined for their ability to undergo genetic exchange with exogenous L1 sequences. The exogenous L1 sequences, carried by a shuttle vector, consisted of an internal fragment from L1Md-A2, a previously described member of the L1 family of the mouse. Using an assay that does not require the reconstitution of a selectable marker we found that this vector, in either circular or linear form, acquired DNA sequences from endogenous L1 elements at a frequency of 10(-3) to 10(-4) per rescued vector. Physical analysis of the acquired L1 sequences revealed that distinct endogenous L1 elements acted as donors and that different subfamilies participated. These results demonstrate that L1 elements are readily capable of genetic exchange. Apart from gene conversion events, the acquisition of L1 sequences outside the region of homology suggested that a second mechanism was also involved in the genetic exchange. A model which accounts for this mechanism is presented and its potential implication on the rearrangement of L1 elements is discussed.