A transposon-like structure in the 5'' flanking sequence of a legumin gene from Pisum sativum

Summary The legumin storage proteins of Pisum sativum are coded for by a multigene family. An insertion element (Pis1) has been found integrated into the 5 flanking sequence of the legC legumin seed storage protein gene. This element contains all the sequence features of the CACTA family of insertion elements, has perfect 12 bp inverted repeats at its termini, and generates a target host site duplication upon integration. An 8 bp sequence within the left arm of the insertion element shows perfect homology to a sequence in the legC flanking region. Three stem-loop structures which can be formed within the element have the same stem sequence.     

Strand invasion and DNA synthesis from the two 3' ends of a double-strand break in Mammalian cells

Analysis of the crossover products recovered following transformation of mammalian cells with a sequence insertion ("ends-in") gene-targeting vector revealed a novel class of recombinant. In this class of recombinants, a single vector copy has integrated into an ectopic genomic position, leaving the structure of the cognate chromosomal locus unaltered. Thus, in this respect, the recombinants resemble simple cases of random vector integration. However, the important difference is that the two paired 3' vector ends have acquired endogenous, chromosomal sequences flanking both sides of the vector-borne double-strand break (DSB). In some cases, copying was extensive, extending >16 kb into nonhomologous flanking DNA. The results suggest that mammalian homologous recombination events can involve strand invasion and DNA synthesis by both 3' ends of the DSB. These DNA interactions are a central, predicted feature of the DSBR model of recombination.     

Unequal meiotic crossover: a frequent cause of NF1 microdeletions

Neurofibromatosis type 1 is a common autosomal dominant disorder caused by mutations of the NF1 gene on chromosome 17. In only 5%-10% of cases, a microdeletion including the NF1 gene is found. We analyzed a set of polymorphic dinucleotide-repeat markers flanking the microdeletion on chromosome 17 in a group of seven unrelated families with a de novo NF1 microdeletion. Six of seven microdeletions were of maternal origin. The breakpoints of the microdeletions of maternal origin were localized in flanking paralogous sequences, called "NF1-REPs." The single deletion of paternal origin was shorter, and no crossover occurred on the paternal chromosome 17 during transmission. Five of the six cases of maternal origin were informative, and all five showed a crossover, between the flanking markers, after maternal transmission. The observed crossovers flanking the NF1 region suggest that these NF1 microdeletions result from an unequal crossover in maternal meiosis I, mediated by a misalignment of the flanking NF1-REPs.     

The mechanism of extrachromosomal homologous DNA recombination in plant cells

Summary By cotransfecting plasmids carrying particular mutations in the -glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.     

FB elements are the common basis for the instability of the wDZL and wC Drosophila mutations

The DNA insertions that cause the highly unstable mutations wC and wDZL share extensive homology with the FB family of transposable elements. FB elements carry long, internally repetitious, inverted terminal repeats and thus differ in structure from other transposable elements. Our results suggest that FB elements may excise and cause chromosomal rearrangements at unusually high frequencies. The wC insertion is a single FB element. The wDZL insertion differs in that it contains two FB elements, one at each terminus. The wC and wDZL insertions contain 4.0 and 6.5 kilobase nonhomologous segments between their terminal repeats. In contrast to the middle repetitive FB elements, the central segment of the wDZL insertion is single-copy and present at a fixed location in the wild-type genome. It has apparently been transposed by the action of flanking FB elements, causing the wDZL mutation at its new location.     

Polymerase chain reaction of genes flanked by short noncontiguous sequence motifs

Short DNA sequence motifs have been demonstrated to interact with DNA binding proteins and regulate flanking genes. The short nature and the lack of continuity of many of these DNA binding sites make it difficult to develop an approach to characterize genes that have the same flanking sequences. We tested various oligonucleotide combinations using an immunoglobulin variable region gene family as a model amplification system. One successful amplification strategy used an oligonucleotide containing two known noncontiguous short sequences connected by random insertion of all four bases to maintain the appropriate spacing. A second approach used an oligonucleotide having a single short homologous sequence with the addition of all four bases randomly placed at the 5' end to increase the extent of homology. Both strategies will permit the priming of members of a specific gene family, with the two short sequences bridged by all four bases randomly added being more efficient in the amplification process.     

Heterozygous insertions alter crossover distribution but allow crossover interference in Caenorhabditis elegans

The normal distribution of crossover events on meiotic bivalents depends on homolog recognition, alignment, and interference. We developed a method for precisely locating all crossovers on Caenorhabditis elegans chromosomes and demonstrated that wild-type animals have essentially complete interference, with each bivalent receiving one and only one crossover. A physical break in one homolog has previously been shown to disrupt interference, suggesting that some aspect of bivalent structure is required for interference. We measured the distribution of crossovers in animals heterozygous for a large insertion to determine whether a break in sequence homology would have the same effect as a physical break. Insertions disrupt crossing over locally. However, every bivalent still experiences essentially one and only one crossover, suggesting that interference can act across a large gap in homology. Although insertions did not affect crossover number, they did have an effect on crossover distribution. Crossing over was consistently higher on the side of the chromosome bearing the homolog recognition region and lower on the other side of the chromosome. We suggest that nonhomologous sequences cause heterosynapsis, which disrupts crossovers along the distal chromosome, even when those regions contain sequences that could otherwise align. However, because crossovers are not completely eliminated distal to insertions, we propose that alignment can be reestablished after a megabase-scale gap in sequence homology.     

Directional ligation of long-flanking homology regions to selection cassettes for efficient targeted gene-disruption in Candida albicans

PCR-product directed gene disruption with a marker cassette having short homology regions is often used in Candida albicans. However, it is quite inefficient due to the high frequency of non-homologous recombination at non-targeted loci, which necessitates extensive screening to identify the correct disruptants. Thus, many PCR-based methods to introduce long flanking homology regions have been developed to increase the frequency of integration at the targeted loci. However, these methods are not that amenable for use with the widely employed C. albicans marker cassettes having direct repeats, as these repeats tend to recombine during PCR, resulting in shorter amplified products without the selection marker. To circumvent this limitation, we have developed a dinucleotide-sticky-end-ligation strategy to add one flanking homology region to one side of the selection cassette, and the other flanking homology region to the other side of the selection cassette. This method involves release of the selection cassette from the plasmid by digestion with two different restriction enzymes, followed by partial fill-in, to provide a unique two base overhang at each end of the cassette. The flanking homology regions, corresponding to the gene to be disrupted, are individually PCR-amplified, and treated with T4-DNA Polymerase in the presence of appropriate dNTPs to yield two base-5' overhangs. The primers used for the PCR have additional bases at the 5' ends such that after T4 DNA Polymerase treatment, the two flanks will have distinct overhangs compatible with the overhangs of the partially filled-in selection cassette. The selection cassette and the flanks are then ligated together and directly used to transform C. albicans. We have successfully used this method for disruption of several C. albicans genes. We have also used this method to recreate insertion mutations obtained with transposons to reconfirm the mutant phenotypes. This approach can be extended to other organisms like Schizosaccharomyces pombe which also require long flanking regions of homology for targeted gene disruption.     

A plasmid rescue technique for the recovery of plant DNA disrupted by T-DNA insertion

Arabidopsis mutants generated by insertion of the T-DNA from Ti plasmid 3850∶1003 serve as a starting point for the isolation of novel genes. The disrupted plant DNA can be recovered using a plasmid rescue technique utilizing high efficiency electroporation. Rescued plasmids are resistant to ampicillin and contain an origin of replication from pBR322. Plasmids generated from either the left or right border of the T-DNA that carry flanking DNA sequences can be identified by analyzing the products of restriction enzyme digests on agarose gels. The plasmids with flanking sequences can then serve as a starting point for cloning plant sequences that share homology to the DNA at the point of T-DNA insertion.     

Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells.

We have used DNA-mediated gene transfer to study homologous recombination in cultured mammalian cells. A family of plasmids with insertion and deletion mutations in the coding region of the herpes simplex type 1 thymidine kinase (tk) gene served as substrates for DNA-mediated gene transfer into mouse Ltk- cells by the calcium phosphate technique. Intermolecular recombination events were scored by the number of colonies in hypoxanthine-aminopterin-thymidine selective medium. We used supercoiled plasmids containing tk gene fragments to demonstrate that an overlap of 62 base pairs (bp) of homologous DNA was sufficient for intermolecular recombination. Addition of 598 bp of flanking homology separated from the region of recombination by a double-strand gap, deletion, or insertion of heterologous DNA increased the frequency of recombination by 300-, 20-, or 40-fold, respectively. Linearizing one of the mutant plasmids in a pair before cotransfer by cutting in the area of homology flanking a deletion of 104 bp or an insertion of less than 24 bp increased the frequency of recombination relative to that with uncut plasmids. However, cutting an insertion mutant of greater than or equal to 24 bp in the same manner did not increase the frequency. We show how our data are consistent with models that postulate at least two phases in the recombination process: homologous pairing and heteroduplex formation.     

Nucleotide sequence of a secondary attachment site for bacteriophage lambda on the Escherichia coli chromosome.

The nucleotide sequence of a secondary attachment site for bacteriophage lambda was determined in a region near the rrnB gene at 88 min on the E. coli chromosome. The sequence has a 8 base pair interrupted homology GCT TTTTA to the common core of the primary attachment site (attB) and the corresponding phage sequence (attP). The site of crossover during integration lies probably between nucleotides -3 and +1. The flanking regions have no obvious homology to the arms of either attP or attB.     

Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9

Background

Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired.

Results

We generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17–0.45%) that was used to knock in an approximately 5-kb long DNA fragment.

Conclusion

We developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.

     

The length of homology required for gene targeting in embryonic stem cells.

Homologous recombination has been used to introduce site-specific mutations into murine embryonic stem (ES) cells with both insertion and replacement vectors. In this study, we compared the frequency of gene targeting with various lengths of homology and found a dramatic increase in targeting with an increase in homology from 1.3 to 6.8 kb. We examined in detail the relationship between the length of homology and the gene-targeting frequency for replacement vectors and found that a critical length of homology is needed for targeting. Adding greater lengths of homology to this critical length has less of an effect on the targeting frequency. We also analyzed the lengths of homology necessary on both arms of the vector for gene replacement events and found that 472 bp of homology is used as efficiently as 1.2 kb in the formation and resolution of crossover junctions.     

Minimal extent of homology required for completion of meiotic recombination in Saccharomyces cerevisiae.

The minimal length of contiguous homology required for successful completion of meiotic recombination was investigated by using heterologous insertions to delimit homologous segments of chromosome III in the yeast Saccharomyces cerevisiae. Constructs created in vitro by insertion of selectable markers into the LEU2 locus were transplaced into haploid strains, which were then mated to create diploids containing pairs of insertion heterologies at various distances. Analysis of the meiotic products from these diploids revealed a gradient in the frequency of both reciprocal and nonreciprocal recombination declining monotonically from the 5' end of LEU2. Both types of event were found to be restricted by the presence of the insertion heterologies. The spo13 single division meiosis was exploited to develop a plating assay in which LEU2 diploid spores containing reciprocally recombinant strands derived from events occurring completely within the interval flanked by the insertion heterologies were selected by random spore methods. Reciprocal recombination frequencies measured with this assay decreased linearly with extent, extrapolating to a minimal homology requirement of 150-250 nucleotides. When homology was most severely restricted, unexpected flanking marker configurations among reciprocal recombinants within LEU2 demonstrated the occurrence of complex recombination events. In addition to detecting reciprocal recombinants, the system is capable of measuring the probability that a non-reciprocal recombination event will have one end-point between the heterologous inserts and the other lying outside the interval. The minimal length of homology required for this aspect of recombination was found to be 25-60 nucleotides.     

Meiotic recombination profiling of interspecific hybrid F1 tomato pollen by linked read sequencing

Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non‐coding regions in the gene promoter and 5′UTR regions of genes. Poly‐A/T and AT rich motifs were found enriched in 1 kb promoter regions flanking the CO sites. Non‐crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non‐homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated.     

Distribution, sequence homology, and homing of group I introns among T-even-like bacteriophages: evidence for recent transfer of old introns

Self-splicing group I introns are being found in an increasing number of bacteriophages. Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG). The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations. We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns. A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a "full set" of these introns. Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages. The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations. Several of the introns can home to closely related intronless phages during mixed infections. However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site. The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing. These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages.     

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.     

Construction and characterization of deletions with defined end points in Drosophila using P elements in trans

We used P-element transposase-mediated "male recombination" between two P elements in trans to create genetic deletions that removed a number of loci, including the gene encoding the neuropeptide crustacean cardioactive peptide (CCAP). Two classes of recombinant chromosomes were produced. Approximately one-quarter were viable when homozygous or hemizygous, whereas the remaining lines caused homozygous and hemizygous lethality. Preliminary analyses using PCR and CCAP immunohistochemistry suggested that, whereas the DNA of the viable lines was largely intact, most lethal lines contained chromosomal deletions that were roughly bounded by the insertion sites of the two P elements used. Southern blot analyses of select lethal lines showed that the DNA flanking the deletion was indeed grossly intact whereas the intervening DNA could not be detected. Sequencing across the deletion in three of these lethal lines identified a single line bearing intact genomic DNA on either side of the deletion separated by 30 bp of P-element DNA. The method described here suggests a simple procedure for creating deletions with defined end points. Importantly, it can use preexisting P-element insertion strains and does not rely on the use of transposable elements that are engineered to cause specific DNA rearrangements.     

Structure of the SAD mutation and the location of control sites at silent mating type genes in Saccharomyces cerevisiae.

The SAD mutation, an extra mating type cassette, has been shown to arise from an unequal mitotic crossover between the MAT and HMR loci, resulting in the formation of a hybrid cassette and a duplication of the MAT-HMR interval. The SAD cassette contains the "a" information and left-hand flanking regions from the parental HMRa cassette and the right-hand flanking sequences of the parental MAT cassette. This arrangement of flanking sequences causes a leaky but reproducible mating phenotype correlated with a low-level expression of the cassette as measured by RNA blotting. This weak expression is attributed to the loss of one flanking control site normally present at the silent HM storage loci.