Stretched DNA Fibers Research Articles

Telomere Length Measurement by Molecular Combing.

Telomeres are repetitive regions of DNA bound by specialized proteins at the termini of linear chromosomes that prevent the natural chromosome ends from being recognized as DNA double strand breaks. Telomeric DNA is gradually eroded with each round of cell division, resulting in the accumulation of critically short or dysfunctional telomeres that eventually trigger cellular senescence. Consequently, telomere length is indicative of the proliferative capacity of a cell. Multiple methods exist to measure telomere length and telomere content, but a simple and reliable technique to accurately measure individual telomere lengths is currently lacking. We have developed the Telomere length Combing Assay (TCA) to measure telomere length on stretched DNA fibers. We used TCA to measure telomere erosion in primary human fibroblasts, and to detect telomere lengthening in response to activation of telomere maintenance pathways. TCA was also used to accurately measure telomere length in healthy individuals, and to identify critically short telomeres in patients with telomere biology disorders. TCA is performed on isolated DNA, negating the need for cycling cells. TCA is amenable to semi-automated image analysis, and can be fully automated using the Genomic Vision molecular combing platform. This not only precludes sampling bias, but also provides the potential for high-throughput applications and clinical development. TCA is a simple and versatile technique to measure the distribution of individual telomere lengths in a cell population, offering improved accuracy, and more detailed biological insight for telomere length measurement applications.

Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes “replicative stress” that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.

Analysis of DNA Replication in Fission Yeast by Combing.

DNA replication studies based on population experiments give an average estimate of replication kinetics from many cells. This average replication profile masks the stochastic nature of origin firing in eukaryotes, which is revealed by using single-molecule techniques, such as DNA combing. The analysis of replication kinetics by DNA combing involves isolating DNA from cells that have been pulse-labeled with thymidine analogs and stretching it on a silanized coverslip. The analog-labeled patches on the stretched DNA fibers can then be detected using fluorescent antibodies against the analog. Each fiber represents a part of the genome from a single cell; therefore, it is possible to study the variation in behavior of individual origins from one cell to another. Furthermore, each DNA fiber is uniformly stretched, making it possible to measure distances accurately at kilobase resolution. It is also possible to stretch a high density of fibers on coverslips enabling quantitative data collection.

Digital Determination of Chemotherapeutic Drug‐Induced Damage Sites in Single DNA Molecules using Nanofluidic Channels

We have shown that DNA can be extended on glass slides permitting the observation of fluorescently labeled DNA fibers and facilitating the quantitation of their length. When the stretched DNA fibers are also immunostained for the presence of apurinic/apyrimidinic (AP) sites, it was possible to quantitate the number of AP sites per unit length of DNA. Measurements of AP site frequency that are determined in this manner are comparable in accuracy to the “gold standard” method of AP site quantitation, but have a lower variance and the ability to analyze even single DNA molecules. Using this method, it has been possible to detect the increased frequency of AP site formation in DNA of cells exposed to hydrogen peroxide in cell culture. Furthermore, it was possible to show that DNA located at replication sites were more intensely damaged than the remainder of the DNA. Similar increases in the quantity of DNA damage sites have also been found in the DNA of cells treated with the cancer chemotherapeutic drug doxycycline. While this technique appears to be a powerful method to detect DNA damage in cells exposed to damaging agents in culture and in vivo, it has the liability of being extremely labor intensive and requires an extended time (weeks to days) for completion. To remedy these shortcomings and to allow clinical translation of this technology, we have developed an alternate approach, which involves the extraction of DNA from circulating tumor cells isolated from whole blood using a unique microfluidic device, immunostaining the DNA and the associated DNA AP sites in micro‐scale reaction chambers and the alignment and deposition of individual DNA fibers in nanochannels (<100 nm in width and depth). This enables single DNA molecules to be stretched reproducibly in nanochannels to near their full contour length which facilitates quantification of labeled AP sites. The DNA fibers positioned within the nanochannels are fluorescently imaged and the images subjected to a unique image analysis technique to provide highly quantitative information on AP site frequency. When using this image analysis approach, it is possible to obtain statistically significant results from even single cells. The combination of these microfluidic and nanofluidic devices into a single analysis platform for quantitating rates of DNA damage reduces the time required for this measurement to an hour or less, reduces technical time to that needed to load samples and reagents, and should permit these analyses to be made in the clinical setting by clinical laboratory personnel.Support or Funding InformationSupported by NIH Grants R21‐125337 and P41‐EB020594.

Detection of Transgenes on DNA Fibers.

Fluorescence in situ hybridization (FISH) was developed for detecting specific DNA sequences directly on mitotic or meiotic chromosomes. However, the resolution of FISH on chromosomes is limited by condensed structure of chromatin, and it is difficult to differentiate two target sites close to each other. To overcome this issue, the objects was changed to stretched DNA fibers, and this fiber FISH technique has now been used for revealing genome structure at molecular level. Hybridization and detection procedures of fiber FISH are common with FISH on chromosomes. Therefore, application of fiber FISH is not difficult for the researchers of some experience in ordinary FISH. DNA fibers can be released from nuclei fixed on glass slides using a detergent. The DNA fibers were shred in FISH procedure, and the resultant fragments became small bead-like shape. This makes FISH signals on DNA fibers a series of dots. The size of DNA in the dot is estimated to be approximately 1 kb, it corresponding to the resolution of fiber FISH. This makes it possible to analyze structures of transgenes on DNA fibers in detail.

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian intitiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites.

An Efficient Method of Cotton DNA Fibers Preparation for FISH

Fluorescence in situ hybridization(FISH) has become the most important technique applied in molecular cytogenetics,especially in developing physical maps in plants.As a key technique,FISH on cotton DNA fibers stretched has not been reported yet,possibly owning to the difficulty in their DNA fiber preparation as well as the existence of thick cytoplasm and hard cell walls.Here we present a method of highly efficient preparation of stretched DNA fibers in cotton.Cotton cotyledons germinated in dark moisture chamber for one week were chopped with a sharp sterile scalpel in a Petri dish that contained ice-cold nucleus isolation buffer(MgSO4 10 mmol L?1,KCl 5 mmol L?1,HEPES 0.5 mmol L?1,DTT 1 mg mL?1,Triton X-100 0.25%,PVP40 2%) followed by sequential filtration through 100,50,and 30 μm nylon meshes.Nuclei were obtained by centrifuging the filtrates at 16 000× g for 1 min.Mixture of nucleus lysis buffer(0.5% SDS,5 mmol L-1 EDTA,100 mmol L?1 Tris,pH 7.0) and nuclei was incubated on the slide for 9 min,DNA fibers obtained by dragging and stretching with a clean slide edge from the end to another end on the liquid surface.After incubated at 60℃ overnight,the slides were pretreated with DNase-free RNase and then rinsed in 2×SSC.The probes and DNA fibers were denatured separately,and hybridization mixture was incubated on the slide overnight,followed by post-hybridization rinses in 2×SSC,1×4T.The slides were blocked with 5% BSA and covered with antibody for 1h.After rinsed with 1×TNT the slides were counterstained with 4',6-diamidino-2-phenylin-dole(DAPI) and followed by rinsing in 1×PBS.After mounting the slides with Vectashield mounting medium the hybridization signals were observed under a fluorescence microscope.Images were captured by a charge-coupled device(CCD) system and brought together to make the plate using Adobe Photoshop 7.0 software.The results indicated that it was easier to release nuclei from cells in nucleus isolation buffer by chopping cotyledon,and slowly and smoothly dragging the nuclei solution with a slide edge from the end to another end on slide treated with poly-L-lysine.Highly stretched and intact DNA fibers were obtained.This method is very simple and rapid,which takes only 30 min to finish the entire process,and it is also safe because poisonous mercaptoethanol is replaced by dithiothreitol.The linear or near-linear stretches of beads on-a-string signals with cotton genomic DNA and 45S rDNA as probes showed that the DNA fibers were suitable for FISH.

The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest

Werner syndrome is an autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several lines of evidence have suggested that the Werner syndrome protein WRN plays a role in DNA replication and S-phase progression. In order to define the exact role of WRN in genomic replication we examined cell cycle kinetics during normal cell division and after methyl-methane-sulfonate (MMS) DNA damage or hydroxyurea (HU)-mediated replication arrest following acute depletion of WRN from human fibroblasts. Loss of WRN markedly extended the time cells needed to complete the cell cycle after either of these genotoxic treatments. Moreover, replication track analysis of individual, stretched DNA fibers showed that WRN depletion significantly reduced the speed at which replication forks elongated in vivo after MMS or HU treatment. These results establish the importance of WRN during genomic replication and indicate that WRN acts to facilitate fork progression after DNA damage or replication arrest. The data provide a mechanistic basis for a better understanding of WRN-mediated maintenance of genomic stability and for predicting the outcomes of DNA-targeting chemotherapy in several adult cancers that silence WRN expression.

Interstitial telomeric sequence blocks in constitutive pericentromeric heterochromatin from Pyrgomorpha conica (Orthoptera) are enriched in constitutive alkali-labile sites

The long interstitial telomeric repeat sequence (ITRS) blocks located in the pericentromeric chromosomal regions of most of Chinese hamster chromosomes behave as hot spots for spontaneous and induced chromosome breakage and recombination. The DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) procedure demonstrated that these ITRS are extremely sensitive to alkaline unwinding, being enriched in constitutive alkali-labile sites (ALS). To determine whether this chromatin modification occurs in other genomes with large ITRS that are not phylogenetically related to mammalian species, the grasshopper Pyrgomorpha conica was analyzed. We chose this species because, with conventional FISH, their chromosomes yield extremely small telomeric signals when probed with the (TTAGG) n polynucleotide, but large ITRS blocks as part of their pericentromeric constitutive heterochromatin. A high density of constitutive ALS was evidenced in the ITRS when intact meiotic cells or somatic cells were subjected to the DBD-FISH technique and probed with the specific telomeric DNA. DBD-FISH with simultaneous hybridization using telomeric and whole genome DNA probes showed that the ITRS tend to colocalize with areas of stronger signal from the whole genome probe. Nevertheless, the signal from the whole genome was more widespread than that from the ITRS, thus providing evidence that a high frequency of constitutive ALS was present in more than one DNA sequence type. Furthermore, stretched DNA fibers processed with DBD-FISH, revealed a distribution of telomeric sequences alternating interspersed with other possible highly repetitive DNA sequences. The abundance of ALS varied from one meiotic stage to another. Interestingly, most of the breakage and meiotic recombination in males takes place close to the constitutive heterochromatin, particularly enriched in ALS. These results provide further evidence of a particular, and possible universal, chromatin structure enriched in constitutive ALS at constitutive heterochromatic regions.

Spatial distribution and specification of mammalian replication origins during G1 phase.

We have examined the distribution of early replicating origins on stretched DNA fibers when nuclei from CHO cells synchronized at different times during G1 phase initiate DNA replication in Xenopus egg extracts. Origins were differentially labeled in vivo versus in vitro to allow a comparison of their relative positions and spacing. With nuclei isolated in the first hour of G1 phase, in vitro origins were distributed throughout a larger number of DNA fibers and did not coincide with in vivo origins. With nuclei isolated 1 h later, a similar total number of in vitro origins were clustered within a smaller number of DNA fibers but still did not coincide with in vivo origins. However, with nuclei isolated later in G1 phase, the positions of many in vitro origins coincided with in vivo origin sites without further change in origin number or density. These results highlight two distinct G1 steps that establish a spatial and temporal program for replication.

Construction of a swine artificial chromosome: a novel vector for transgenesis in the pig

A de novo SAC was constructed by making use of YAC technology and a humanized yeast strain. The construct (about 310 kb) contained pig centromeric DNA and the Neo gene. The construct was introduced into a pig cell line by yeast-mammalian cell fusion and G418 resistant clones were obtained. One clone was characterized by FISH and shown to contain an episomally located microchromosome containing YAC, Neo and pig centromere sequences. FISH analysis over time showed that the SAC was mitotically stable for at least 34 generations in the absence of selection. The size of the SAC was determined by confocal microscopy of the SAC and shown to be approximately 7 Mb, which is about 25-fold greater than the size of the original YAC. From its behavior in pulsed field gel electrophoresis, FISH analysis of stretched DNA fibers, and its appearance under scanning confocal microscopy, it was concluded that the SAC is a circularized and multimerized derivative of the original YAC. Possible applications as vectors for animal transgenesis are discussed.

Identification of duplicated genes in 17q11.2 using FISH on stretched chromosomes and DNA fibers.

Recent evidence has been provided that links duplicons (REP-P and REP-M) in 17q11.2 flanking the neurofibromatosis type 1 (NF1) gene to the breakpoints of the NF1 microdeletion syndrome. The physical mapping and structural definition of duplicated regions is often impossible by conventional approaches, and so we have used high resolution fluorescence in situ hybridization (FISH) with locus-specific probes of limited size on chromosomes stretched to different degrees to identify novel duplicated genes and expressed sequence tags (ESTs) mapping to 17q11.2. This approach has allowed us to detect and map duplications of the BLMH and GOS28 genes, with one copy lying centromeric and one telomeric to the NF1 gene, and an SHGC30113 transcript with one copy being adjacent and the other distal to the NF1 3' untranslated region. Double-color FISH with a BLMH-specific probe on stretched chromosomes showed that the telomeric BLMH repeat lacked the 5' end of the gene and was 0.8 Mb from its centromeric copy. The distance between the SHGC30113 repeats was estimated as being 500 kb by double-color FISH on highly stretched chromosomes and DNA fibers. The latter approach revealed adjacent SHGC30113-BLMH-specific signals relating to the incomplete BLMH copy. The use of FISH on stretched chromosomes and, where applicable DNA fibers, is a powerful tool for identifying and finely characterizing duplicated regions whose mapping by the classical physical mapping approach is often precluded.

Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification.

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Integration of Banana Streak Badnavirus into theMusaGenome: Molecular and Cytogenetic Evidence

Breeding and tissue culture of certain cultivars of bananas (Musa) have led to high levels of banana streak badnavirus (BSV) infection in progeny from symptomless parents. BSV DNA hybridized to genomic DNA of one such parent, Obino l'Ewai, suggesting integration of viral sequences. Sequencing of clones of Obino l'Ewai genomic DNA revealed an interface between BSV andMusasequences and a complex BSV integrant.In situhybridization revealed two different BSV sequence locations in Obino l'Ewai chromosomes and a complex arrangement of BSV andMusasequences was shown by probing stretched DNA fibers. This is the first report of integrated sequences that possibly lead to a plant pararetrovirus episomal infection by a mechanism differing markedly from animal retroviral systems.

Arrangement of individual human ribosomal DNA fragments on stretched DNA fibers.

We studied the arrangement of individual human ribosomal (r)DNA repeats by direct visualization of rDNA sequences. We used high resolution fluorescence in situ hybridization on preparations of DNA fibers released from interphase nuclei of HeLa cells. Probes from both the transcription unit and the intergenic spacer were used, and lengths of signals and of the gaps in between were measured and compared to molecular data. We could visualize the repetitive arrangement of individual rDNA sequences at the single gene level. No inversions or deletions were detected. The intergenic spacer was found to be shorter than expected, indicating a length polymorphism.

High-resolution FISH of stretched chromosome fibers

High resolution fluorescence in situ hybridization (FISH), so-called FIBER-FISH, was first described by Heng et al. (1992) for long stretched DNA-fibers and was later subjected to modifications by other investigators (Fidlerova et al. 1994, Heiskanen et al. 1996). The resolution using cosmids on DNA fibers was restricted by the length of the visible, decondensed DNA strand up to 150 to 450 kb. Using earlier protocols, it was not possible to obtain a coherent DNA strand showing a longer section of cosmids in direct orientation. However, freshly prepared lymphocyte suspension could be used (for lymphocyte preparation see Arnoldus et al., 1990, Verma et al., 1989).This method described here is simple, but very efficient. It allows the presentation of cosmids over a region up to 8 Mb in a linear orientation. Thus, the visualization of the transcriptional orientation of genes is possible within a bigger range. Moreover, the modified treatment allows the use of archival suspensions of cultured lymphocytes and is also available for YACs. The main steps are a special treatment of up to two year old lymphocyte suspensions, the disruption of nuclei by a shortened PBS incubation previous to alkaline treatment and the release of chromosome fibers from interphase nuclei by special handling of the slides. With these modifications, we were able to obtain released chromosome fibers, which were suitable for a high resolution two-color FISH. This method has been specially tested for lymphocyte nuclei (Fuchs et al., 1997).

High-resolution FISH of the entire integrated Epstein-Barr virus genome on extended human DNA.

Here we report a high-resolution fluorescence in situ hybridization (FISH) analysis of the integrated Epstein-Barr virus (EBV) genome in chromosomes, decondensed interphase nuclear chromatin, and linearly extended chromatin fibers. We analyzed the EBV DNA integrated into the human genome in the well-characterized Burkitt's lymphoma cell line Namalwa, which contains two complete EBV genomes. The integration occurs via the terminal repeats of the virus and was always detectable at chromosome band 1p35. Using the biotinylated BamHIW fragment of the viral DNA, we observed distinct pairs of signals or small nuclear RNA "tracks" within interphase nuclei. FISH to stretched DNA fibers has a higher resolving power and; therefore, enables analysis of the structural organization of DNA. Application of this methodology to linearly extended chromatin of Namalwa cells using different EBV fragments allowed us to visualize the ordered arrangement of the integrated virus. Based on the predicted span of 0.34 nm per base pair for relaxed DNA, length measurements of 30 images showed a good correlation between the mean physical length of hybridized EBV DNA of 52.8 microns (158 kb) without the terminal repeats, and the EBV genomic length of 172 kb, including the terminal repeats. This DNA mapping procedure represents a useful tool for studying the structural organization of integrated viral genomes, and its application will have implications for the understanding of integration processes.