High resolution mapping DNAs by R-loop atomic force microscopy.
ABSTRACT: R-loops formed by short RNA transcripts have been imaged by atomic force microscopy (AFM) at a constant force in the height mode. The technique was applied to mapping the human endogenous retrovirus K10 family (HERV-K10) long terminal repeats (LTR) within individual plasmids and cosmids. RNA probes specific for the U3 (384 nt) and U5 (375 nt) LTR regions separated by a span of 200 bp were used for R-loop formation with LTRs located within plasmid (3.8 kb) or cosmid ( approximately 40 kb) DNAs. R-loops stabilized by glyoxal treatment and adsorbed onto the mica surface in the presence of magnesium ions looked like looped out segments of RNA:DNA hybrids. The total yield of R-loops was usually approximately 95%. The RNA:DNA hybrids were found to be 12-15% shorter than the corresponding DNA:DNA duplex. The two regions of the LTR could be easily discerned in the AFM images as clearly separated loops. R-loop positions determined on cosmids by AFM were accurate to approximately 0.5% of the cosmid length. This technique might be easily adapted for mapping various sequences such as gene exons or regulatory regions and for detecting insertions, deletions and rearrangements that cause human genetic diseases.
Project description:<h4>Background</h4>Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination.<h4>Methods</h4>Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection.<h4>Results</h4>We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites.<h4>Conclusions</h4>We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.
Project description:A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification.
Project description:Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.
Project description:We have constructed defined human cytomegalovirus (CMV) mutants by cotransfecting overlapping cosmid clones spanning the 230-kbp genome. Using this strategy, we have introduced a 13-kbp region of DNA from a virulent strain of CMV into a defined position within the avirulent CMV(Towne) genome. Although more than 80% of the genome of these recombinant viruses was derived from Towne DNA, their plaque morphology more closely resembled that of Toledo. To date, CMV is the largest virus and requires the greatest number of cosmids to be regenerated via overlapping cosmid cotransfection.
Project description:DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.
Project description:Serial passage of axenically cultured Leishmania chagasi promastigotes results in a progressive diminution in resistance to complement-mediated lysis (CML), whereas high CML resistance is seen in infectious metacyclic promastigotes from the sandfly vector as well as metacyclic-like promastigotes within low-passage cultures at stationary growth phase. As we previously reported, in a screen seeking to identify novel genes involved in CML resistance: (1) a genomic cosmid library derived from DNA of CML-resistant L. chagasi promastigotes was transfected into high-passage (constitutively CML-sensitive) L. chagasi promastigotes; (2) transformants were screened for acquisition of CML-resistance; (3) multiple cosmid-transfectants exhibited partial CML resistance; and (4) the sequence for one of the cosmids (Cosmid 51) was determined. This report extends the analysis of Cosmid 51, and identifies by deletion analysis a subregion of the cosmid insert that is critical to the CML-resistance phenotype of Cosmid 51 transformants. We also report the sequence determination and initial CML-resistance activity of another cosmid that also confers partial resistance to CML.
Project description:The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.
Project description:R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.
Project description:Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing approximately 5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus (ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met-hepatocyte growth factor receptor. The other cosmid contains only the two short 5'-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the beta-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome.
Project description:Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I-SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.