Replacement synthesis labeling of DNA molecules in vitro using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair

James, C D; Leffak, I.M.

doi:10.1016/0003-2697(84)90421-4

Cited by 17 publications

(6 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The probe was labeled to 1 to 2 dpm/fg by replacement synthesis (18). RNA was isolated by the methods of Chirgwin et al (5) and Glisin et al (12).…”

mentioning

confidence: 99%

Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Trempe¹,

Lindstrom²,

Leffak³

1988

Mol. Cell. Biol.

View full text Add to dashboard Cite

We used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocytes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, we conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction.The replication of eucaryotic chromosomes is under both spatial and temporal controls, as evidenced by the firing of local clusters of replicons according to developmentally regulated programs and the replication of particular chromosome domains during similar intervals of the S phase in successive cell cycles (reviewed in reference 16). That the genomic location of an immunoglobulin gene sequence can influence its schedule of replication has been shown by Calza et al. (3).A relationship between transcriptional activity and the timing of replication has been inferred based on the observation that genes are generally replicated earlier in the S phase in cells where they are transcribed than in cells where they are inactive (7, 13). In the case of the Xenopus laevis kidney cell line TrXo, translocation of oocyte-type 5S rDNA genes from their normal telomeric location to a pericentromeric location is correlated with both a shift from late to early S-phase replication and the accumulation of oocyte 5S transcripts (11, 15), supporting the suggestion that the earlier replication of somatic versus oocyte 5S genes in normal cells may contribute to the selective transcription of the somatic genes (14,38).A structural link between replication and transcription is suggested by the observations that under normal cell growth conditions the Hi histones and octamer cores of bulk chromatin are conservatively replicated (23-25, 34, 36), whereas the core histone octamers of transcribed chromatin and the HMG 14/17 nonhistone pair preferentially associated with transcribed...

show abstract

“…The probe was labeled to 1 to 2 dpm/fg by replacement synthesis (18). RNA was isolated by the methods of Chirgwin et al (5) and Glisin et al (12).…”

mentioning

confidence: 99%

Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Trempe¹,

Lindstrom²,

Leffak³

1988

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…Isolated nuclei were incubated in a standard reaction mixture containing 33 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7 (25). The filters were washed in 0.5x SSC-0.5% sodium dodecyl sulfate at 65°C and exposed to Kodak XR-5 film at -80°C with intensifying screens for 2 to 7 days.…”

mentioning

confidence: 99%

Polarity of DNA replication through the avian alpha-globin locus.

James

Leffak

1986

Mol. Cell. Biol.

View full text Add to dashboard Cite

Toward understanding the controls affecting eucaryotic chromosome replication, we used a runoff replication assay to investigate whether the activity of a gene is related to its use of an upstream or downstream replication origin. When in vivo-initiated DNA polymerases are allowed to complete replication in vitro in the presence of bromodeoxyuridine triphosphate the density label is preferentially incorporated into origin-distal regions of DNA. Isopycnic centrifugation and blot hybridization analysis of the relative bromodeoxyuridine triphosphate incorporation into fragments spanning the chicken alpha-globin locus indicate that this region is replicated from an upstream origin both in chicken lymphocytes and in erythrocytes. Thus the replication polarity of these genes does not change as a function of transcriptional activity, consistent with earlier suggestions that DNA replication in the transcriptional direction may be a necessary but not sufficient condition for gene expression.The ordered nature of eucaryotic chromosome replication is evident in the replication of particular chromosome domains during discrete intervals of the S phase and in the synchronous initiation of replicon clusters whose size and number vary in a tissue-specific and developmentally regulated manner (reviewed in references 16, 21, and 47). These observations are consistent with recent data showing that DNA sequences complementary to probes for individual transcribed genes preferentially replicate early in the S phase and that chromosome position influences the timing of gene replication (6,11,17,20). In Saccharomyces cerevisiae, electron microscopic and biochemical data imply that DNA synthesis initiates at specific loci (5,7,18,46,50); the clearest evidence for the existence of discrete origins in higher eucaryotes comes from studies of the amplification of dihydrofolate reductase genes in CHO cells (22,23) and chorion genes in Drosophila follicle cells (36,44), where the extent -of amplification decreases bidirectionally from a central domain.The apparent nonrandom selection of sites for replication initiation and the asymmetry of chromatin replication at the level of DNA synthesis (16, 35) and protein deposition (28) have contributed to the proposal that replication origin selection may regulate gene activity (42, 49). As an initial step toward testing this hypothesis we have used a runoff replication assay to determine the direction of replication of the chicken alpha-globin genes in cells where these sequences are expressed (erythrocytes) or quiescent (lymphocytes). Following the logic used to locate the simian virus 40 origin of replication (12, 34), isolated nuclei were incubated in a replication cocktail containing bromodeoxyuridine (BrdU) triphosphate (BrdUTP) and allowed to complete the synthesis of DNA chains initiated in vivo. The direction of replication was deduced based on the relative incorporation of BrdU into DNA fragments encompassing the globin locus; those fragments farthest from the origin of replication incorpora...

show abstract

“…Postemulsion library recovery in later rounds with only two hbRCA primers was especially challenging, as PCR recovery reactions were dominated by parasites, small off-target PCR products that were preferentially amplified over the larger (∼1800 bp) polymerase gene product. To circumvent these difficulties, we developed a novel parasite removal strategy that relied on exonuclease III (ExoIII), a synchronous 3′ to 5′ DNA exonuclease with well-studied reaction kinetics. − By lowering the digestion reaction temperature to 25 °C, where ExoIII cuts at a rate of ∼100 bp, we found that 8 min of treatment would completely digest products less than 1700 bp in length while leaving overhangs on larger products. The remaining hemiduplexes served as substrates for primer extension by Taq polymerase at 68 °C (a temperature that also denatured ExoIII).…”

Section: Resultsmentioning

confidence: 82%

Evolution of a Thermophilic Strand-Displacing Polymerase Using High-Temperature Isothermal Compartmentalized Self-Replication

et al. 2018

View full text Add to dashboard Cite

Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions, where the lack of thermal cycling reduces equipment needs and improves the time to answer, especially for point-of-care applications. In order to improve the function of strand-displacing polymerases, we have developed an emulsion-based directed evolution scheme, high-temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand-displacing polymerase variant that performs well in loop-mediated isothermal amplification and rolling circle amplification, even after thermal challenges of up to 95 °C that lead to better primer annealing. The new enzyme (v5.9) is also capable of a variety of new reactions, including isothermal hyperbranched rolling circle amplification. The HTI-CSR method should now prove useful for evolving additional beneficial phenotypes in strand-displacing polymerases.

show abstract

Replacement synthesis labeling of DNA molecules in vitro using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair

Cited by 17 publications

References 10 publications

Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Polarity of DNA replication through the avian alpha-globin locus.

Evolution of a Thermophilic Strand-Displacing Polymerase Using High-Temperature Isothermal Compartmentalized Self-Replication

Contact Info

Product

Resources

About