Recombination between short direct repeats in Streptomyces lavendulae plasmid DNA.

Streptomyces lavendulae S985 carried two plasmids, pSL1 and pSL2. pSL2 contained all of the pSL1 sequences plus a tandem duplication of 900 base pairs from a region of pSL1. Sequence analysis of the duplication junction suggested that the duplication occurred by recombination between short direct repeats of as little as 5 base pairs.     

Recombination between short direct repeats in a RecA host

Summary Spontaneous deletion derivitives of plasmid pHV14, a recombinant of pBR322, have been isolated in the recA strain HB101. About 2.5 kilobases (Kb) of DNA has been lost in each of the four plasmids described including, in two cases the region involved in control of copy number and the initiation of replication.Sequence analysis of each deletion junction has revealed that in 3 out of 4 cases the deletion event occured by recombination between short direct repeats of as little as 7 base pairs (bp).     

Recombination in recA cells between direct repeats of insertion element IS1.

The IS1 sequences that flank the Tn9 chloramphenicol acetyltransferase gene as direct repeats recombine after transformation into an Escherichia coli recA strain. The recombination requires the lambda pL promoter on the plasmid. A plasmid that contains mutant IS1 elements does not recombine. These results indicate that this recombination requires an IS1-specific gene product. The recombinational activity of IS1 may resolve transient cointegrates formed during the transposition of IS1. I discuss a possible role for the lambda pL promoter.     

Identification and nucleotide sequences of two similar tandem direct repeats in Epstein-Barr virus DNA

Epstein-Barr virus DNA is known to have partially homologous segments, designated DL and DR, near the left and right ends of the long unique region (Raab-Traub et al., Cell 22:257-267, 1980). DL and DR are each partially composed of tandem direct repeat sequences. DL contains 11 to 14 repeats of a 124-base-pair sequence designated IR2. DR contains approximately 30 direct repeats of a 103-base-pair sequence designated IR4. The DL and DR sequences have colinear partial homology for approximately 2.4 and 1.5 kilobase pairs to the right of IR2 and IR4, respectively. IR2 and IR4 are similar sequences and evolved in part from a common ancestor. Both sequences are 84% guanine and cytosine and have limited homology to Epstein-Barr virus IR1 and to the herpes simplex virus type 1 inverted terminal repeat "a" sequence. IR2 encodes part of an abundant 2.5-kilobase persistent early EBV RNA expressed in productively infected cells, but does not encode part of the 3-kilobase Epstein-Barr virus RNA which is transcribed from the adjacent IR1-U2 region of the Epstein-Barr virus genome in latently infected cells.     

Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA

Large-scale deletions of mitochondrial DNA (mtDNA) have been described in patients with progressive external ophthalmoplegia (PEO) and ragged red fibers. We have determined the exact deletion breakpoint in 28 cases with PEO, including 12 patients already shown to harbor an identical deletion; the other patients had 16 different deletions. The deletions fell into two classes. In Class I (9 deletions; 71% of the patients), the deletion was flanked by perfect direct repeats, located (in normal mtDNA) at the edges of the deletion. In Class II (8 deletions; 29% of patients), the deletions were not flanked by any obviously unique repeat element, or they were flanked by repeat elements which were located imprecisely relative to the breakpoints. Computer analysis showed a correlation between the location of the deletion breakpoints and sequences in human mtDNA similar to the target sequence for Drosophila topoisomerase II. It is not known how these deletions originate, but both slipped mispairing and legitimate recombination could be mechanisms playing a major role in the generation of the large mtDNA deletions found in PEO.     

Recombination between two 14-bp homologous sequences as the mechanism for gene deletion in factor IX Seattle 1.

Factor IXSeattle 1 is a 10-kb intragenic deletion identified in a family that has hemophilia B. By sequencing across the site of the deletion, we discovered at the deletion junction a 13-bp sequence (5' . . . TAGAA-GTTCACTT . . . 3') that was homologous to two 14-bp sequences 10 kb apart in introns D and F of the normal factor IX gene. The presence of these homologous sequences in two different regions of the normal gene allows us to propose that genetic recombination has occurred between the sequences, resulting in the gene deletion. The precise recombination site was able to be localized to one of 5 bp (5' . . . AGTTC . . . 3') in the middle of the homologous sequences. The exact length of the deletion is 10,000 bp.     

Phylogenetic relationships between adenoviruses as inferred from nucleotide sequences of inverted terminal repeats

The nucleotide (nt) sequences of inverted terminal repeats (ITR) from human adenovirus (Ad) 19, bovine Ad1 (BAd1), bovine Ad3 (BAd3), canine Ad2 (CAd2) and an avian Ad, EDS-76, were determined. The length of the ITR sequence was 160 bp in Ad19, 159 bp in BAd1, 195 bp in BAd3, 196 bp in CAd2 and 52 bp in EDS-76. CAd2 had the longest ITR among the examined Ads, BAd3 the second longest, and EDS-76 had the shortest ITR. A TAAT sequence located between the 10th and 13th nt counted from the ends was conserved in all Ads examined so far. To determine phylogenetic relationships among human and animal Ads, sequences of their ITRs were compared, and a phylogenetic tree was constructed by using the maximum-likelihood method. It is the method involving statistical analysis of computing the probability of a particular set of sequences on a given tree and maximizing this probability over all evolutionary trees [Felsenstein, J. Mol. Evol. 17 (1981) 368-376]. From these analyses, it was found that members belonging to the same human Ad subgenus are related closely to each other, whereas representatives of different human subgenera are distributed rather divergently among animal Ads.     

Recombination between repeated DNA sequences occurs more often in plasmids than in the chromosome of Bacillus subtilis

Summary Directly repeated pBR322 sequences 3.7–3.8 kb long recombine with a frequency of about 10% per generation when carried on plasmids related to pC194 and 0.01% per generation when carried on the Bacillus subtilis chromosome. Recombination is therefore 1,000 times more efficient in plasmids than in the chromosome of this organism.     

Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences.

A cell-free system of nuclear extracts from BHK21 cells has been developed to catalyse recombination in vitro between the DNA of adenovirus type 12 (Ad12) and two different hamster preinsertion sequences. The pBR322 cloned 1768 bp fragment p7 and the 3.1 kbp fragment p16 from BHK21 hamster DNA had previously been identified as the preinsertion sites corresponding to the junctions between Ad12 DNA and hamster DNA in cell line CLAC1 and in the Ad12-induced tumour T1111(2), respectively. Preinsertion sequences, which had recombined previously with foreign (Ad12) DNA, might again be recognized by the recombination system even in a cell-free system. PstI cleaved Ad12 DNA and the circular or the EcoRI linearized p7 or p16 preinsertion sequences were incubated with nuclear extracts. Recombinants were isolated by transfecting the DNA into recA- Escherichia coli strains and by screening for Ad12 DNA-positive colonies. Without a selectable eukaryotic marker, all Ad12 DNA positive recombinants were registered. Out of a total of greater than 90 p7-Ad12 DNA recombinants, 21 were studied by restriction-hybridization, and four by partial nucleotide sequence analyses. Among the p16-Ad12 DNA recombinants, four were analysed. The sites of linkage between Ad12 DNA and p7 or p16 hamster DNA were all different and distinct from the original CLAC1 or T1111(2) junction site between Ad12 and hamster DNA. The in vitro recombinants were not generated by simple end-to-end joining of the DNA fragments used in the reaction but by genetic exchange. Thirteen of the 25 recombinants were derived from the 61-71 map unit fragment of Ad12 DNA. Recombination experiments between Ad12 DNA and four randomly selected unique or repetitive hamster DNA sequences of 1.5-6.2 kbp in length did not yield recombinants. Apparently, the p7 and p16 hamster preinsertion sequences recombined with Ad12 DNA with a certain preference.     

Variable maternal methylation overlapping the nc886/vtRNA2-1 locus is locked between hypermethylated repeats and is frequently altered in cancer

Cancer is as much an epigenetic disease as a genetic one; however, the interplay between these two processes is unclear. Recently, it has been shown that a large proportion of DNA methylation variability can be explained by allele-specific methylation (ASM), either at classical imprinted loci or those regulated by underlying genetic variants. During a recent screen for imprinted differentially methylated regions, we identified the genomic interval overlapping the non-coding nc886 RNA (previously known as vtRNA2-1) as an atypical ASM that shows variable levels of methylation, predominantly on the maternal allele in many tissues. Here we show that the nc886 interval is the first example of a polymorphic imprinted DMR in humans. Further analysis of the region suggests that the interval subjected to ASM is approximately 2 kb in size and somatically acquired. An in depth analysis of this region in primary cancer samples with matching normal adjacent tissue from the Cancer Genome Atlas revealed that aberrant methylation in bladder, breast, colon and lung tumors occurred in approximately 27% of cases. Hypermethylation occurred more frequently than hypomethylation. Using additional normal-tumor paired samples we show that on rare occasions the aberrant methylation profile is due to loss-of-heterozygosity. This work therefore suggests that the nc886 locus is subject to variable allelic methylation that undergoes cancer-associated epigenetic changes in solid tumors.     

Variable maternal methylation overlapping the nc886/vtRNA2-1 locus is locked between hypermethylated repeats and is frequently altered in cancer

Cancer is as much an epigenetic disease as a genetic one; however, the interplay between these two processes is unclear. Recently, it has been shown that a large proportion of DNA methylation variability can be explained by allele-specific methylation (ASM), either at classical imprinted loci or those regulated by underlying genetic variants. During a recent screen for imprinted differentially methylated regions, we identified the genomic interval overlapping the non-coding nc886 RNA (previously known as vtRNA2-1) as an atypical ASM that shows variable levels of methylation, predominantly on the maternal allele in many tissues. Here we show that the nc886 interval is the first example of a polymorphic imprinted DMR in humans. Further analysis of the region suggests that the interval subjected to ASM is approximately 2 kb in size and somatically acquired. An in depth analysis of this region in primary cancer samples with matching normal adjacent tissue from the Cancer Genome Atlas revealed that aberrant methylation in bladder, breast, colon and lung tumors occurred in approximately 27% of cases. Hypermethylation occurred more frequently than hypomethylation. Using additional normal-tumor paired samples we show that on rare occasions the aberrant methylation profile is due to loss-of-heterozygosity. This work therefore suggests that the nc886 locus is subject to variable allelic methylation that undergoes cancer-associated epigenetic changes in solid tumors.     

Plasmid deletion formation between short direct repeats in Bacillus subtilis is stimulated by single-stranded rolling-circle replication intermediates

Summary The effects of the rolling-circle mode of replication and the generation of single-stranded DNA (ss DNA) on plasmid deletion formation between short direct repeats in Bacillus subtilis were studied. Deletion units consisting of direct repeats (9, 18, or 27 bp) that do or do not flank inverted repeats (300 bp) were introduced into various plasmid replicons that generate different amounts of ss DNA (from 0% to 40% of the total plasmid DNA). With ss DNA-generating rolling-circle-type plasmids, deletion frequencies between the direct repeats were 3- to 13-fold higher than in plasmids not generating ss DNA. When the direct repeats flanked inverted repeats the deletion frequencies in ss DNA-generating plasmids were increased by as much as 20- to 140-fold. These results support models for deletion formation based on template-switching errors during complementary strand synthesis of rolling-circle-type plasmids. The structural instability (deletion formation between short direct repeats) of the ss DNA-generating plasmid pTA1060 in B. subtilis was very low in the presence of a functional initiation site for complementary strand synthesis (minus origin). This observation suggests that it will be possible to develop stable host-vector cloning systems for B. subtilis.     

Pig genome analysis: differential distribution of SINE and LINE sequences is less pronounced than in the human and mouse genomes

The distribution of SINE and LINE sequences in the pig genome was examined by fluorescence in situ hybridization (FISH), interspersed repeat PCR, and restriction analysis of high molecular weight DNA. FISH revealed a largely uniform hybridization to the euchromatic chromosome regions with both interspersed repeats, although a bias toward the G-bands was observed for the LINE probe. Southern blots of inter-SINE and inter-LINE PCR products showed strong hybridization to LINE and SINE probes, respectively. High molecular weight DNA derived from a pig |m~ hamster hybrid cell line was cut with a panel of G + C and A + T rich rare cutter restriction enzymes, then run on a pulsed field gel and Southern blotted. Sequential hybridization with SINE and LINE probes showed that SINE hybridization was to relatively low molecular weight fragments with the G + C rich enzymes, whereas the LINE probe gave hybridization to significantly larger fragments produced by these enzymes. DNA samples digested with A + T rich enzymes gave essentially similar patterns with SINE and LINE probes. We conclude that the pattern of differential distribution of SINEs and LINEs, which has been described in man and mouse, does exist in the pig but is much less pronounced. Received: 25 April 1995 / Accepted: 1 September 1995     

Looking for relationships between the most repeated dispersed DNA sequences in the mouse: small R elements are found associated consistently with long MIF repeats.

Four highly-repeated dispersed DNA sequence families have been described in the mouse genome. These are the three small elements B1, B2 and R and the large (6 kb) MIF element. Together these comprise approximately 10% of the mouse genome. Possible relationships between these families are pertinent to the genome as a whole. We report here that the B1s, B2s and Rs are all randomly organized in the genome with respect to each other. Surprisingly though, the R and MIF families are found together consistently in a set of random genomic clones and in selected clones. We find Rs often located on one end of the MIF at a consistent site and conclude that a minority of Rs are an integral part of MIF while the majority of Rs are not associated with MIFs. We propose that isolated R elements are truncated forms of MIF. Also we speculate on the mechanism of dispersal of these elements through the mouse genome.     

The effect of the length of direct repeats and the presence of palindromes on deletion between directly repeated DNA sequences in bacteriophage T7.

The frequency of genetic deletion between directly repeated DNA sequences in bacteriophage T7 was measured as a function of the length of the direct repeat. The non-essential ligase gene (gene 1.3) of bacteriophage T7 was interrupted with pieces of synthetic DNA bracketed by direct repeats of various lengths. Deletion of these 76 bp long inserts was too low to be measured when the direct repeats were less than 6 bp long. However, the frequency of deletion of inserts with longer direct repeats increased exponentially as the length of the repeats increased from 8 to 20 bp. When inverted repeats (palindromes) were designed in the midst of the insert there was essentially no increase in deletion frequency between 10 bp direct repeats. But, the same palindromic sequences increased the deletion frequency between 5 bp direct repeats by at least two orders of magnitude. Thus, in this system homology at the endpoints is a more important determinant of deletion frequency than is the presence of palindromes between the direct repeats.     

Unequal crossing over as the pathway of adaptive homologous recombination between the direct DNA repeats in tandem duplications of Escherichia coli

Homologous recombination between direct DNA repeats within the extended tandem duplications in E. coli results from unequal sister-chromosome exchanges. This conclusion follows from the observations on the segregation of completely or partly homozygous diploid segregants by heterozygous duplications. The formation of diploid segregants with preserved heterozygosity for the unselected markers could also result from "symmetrical" intrachromosomal recombination. Analysis of the segregant genotypes, however, confirmed their formation via unequal crossing over. The data obtained indicated that in tandem duplications segregation of diploid recombinants of different types was preceded by the formation of triplications as the products of unequal sister-chromosome exchanges. In heterozygous duplications, unequal crossing over is manifested as a highly frequent adaptive exchange, providing the survival of the most part of the duplication-carrying cells on selective medium. It is suggested that adaptive mutagenesis can be the consequence of unequal sister crossing over.