Pool Of DNAs Research Articles

The nuclear location and chromatin organization of active chorion amplification origins.

It remains unclear how certain regions on metazoan chromosomes are selected to initiate DNA replication. In recent years a number of origins of DNA replication have been mapped, but there is still no DNA consensus for predicting where replication will initiate. Evidence suggests that the higher order structure of the nucleus and chromosome influences origin activity. Chromosomal DNA replication is proposed to occur in special compartments in the nucleus called replication foci. Foci in different regions of the nucleus initiate replication at different times of S-phase, suggesting nuclear position may contribute to where and when replication begins. Here we test the contribution of nuclear compartments for well-defined origins, those involved in amplification of the chorion (eggshell) genes during Drosophila oogenesis. The results of three-dimensional confocal microscopy indicate that chorion DNA replication origins are highly active in diverse positions within the nucleus. We also find that chorion replication origins inserted at ectopic chromosomal sites can amplify highly in diverse nuclear locations distinct from the endogenous loci, including when they are buffered against genomic position effects. We used fluorescence in situ hybridization to analyze chromosome structure during amplification. Contrary to the replication factory model, we find no evidence for spooling of DNA toward a replication center. We discuss the implications of these results for understanding the role of higher order structure in amplification and chromosome duplication.

In Vitro Selection of Nonnatural Ribozyme-Catalyzing Porphyrin Metalation

A new macromolecular catalyst, which is composed of nonnatural ribonucleotide, was synthesized by the in vitro selection (SELEX) method. A pool of RNAs consisting of random sequences was obtained by transcription from a pool of synthetic random-sequence DNAs in the presence of 2'-aminocytidine triphosphate (2'-amino-CTP) instead of CTP. The pool was incubated with N-methylmesoporphyrin-immobilized gel; bound nonnatural RNAs were then collected and amplified by reverse-transcription following the polymerase chain reaction. The amplified DNAs were again transcribed in the presence of 2'-amino-CTP and applied to the gel. This selection process was repeated 10 times. The selected RNAs were cloned and sequenced. The RNAs not only bound to the ligand, N-methylmesoporphyrin but also catalyzed the metalation reaction of porphyrin.

In vitro selection of deoxyribozymes with DNA capping activity.

In vitro selection was used to isolate a series of deoxyribozymes from a pool of random-sequence DNAs that catalyze an ATP-dependent self-capping reaction. Each deoxyribozyme catalyzes the transfer of the nucleoside and alpha-phosphate moieties of ATP to the phosphate group located at its 5' terminus, thereby creating a 5',5'-pyrophosphate cap. This same pyrophosphate cap structure is formed by T4 DNA ligase during the classical process of DNA ligation. These DNA capping enzymes representative of a collection of self-processing deoxyribozymes that can be used for the directed modification of DNA.

Isolation of a fluorophore-specific DNA aptamer with weak redox activity.

In vitro selection experiments with pools of random-sequence nucleic acids have been used extensively to isolate molecules capable of binding specific ligands and catalyzing self-modification reactions. In vitro selection from a random pool of single-stranded DNAs has been used to isolate molecules capable of recognizing the fluorophore sulforhodamine B with high affinity. When assayed for the ability to promote an oxidation reaction using the reduced form of a related fluorophore, dihydrotetramethylrosamine, a number of selected clones show low levels of catalytic activity. Chemical modification and site-directed mutagenesis experiments have been used to probe the structural requirements for fluorophore binding. The aptamer recognizes its ligand with relatively high affinity and is also capable of binding related molecules that share extended aromatic rings and negatively charged functional groups. A guanosine-rich single-stranded DNA is capable of binding fluorophores with relatively high affinity and of weakly promoting a multiple-turnover reaction. A simple motif consisting of a three-tiered G-quartet stacked upon a standard Watson-Crick duplex appears to be responsible for this activity. The corresponding sequence might provide a useful starting point for the evolution of novel, improved deoxyribozymes that generate fluorescent signals by promoting multiple-turnover reactions.

In Vitro Selection of a DNA Aptamer Binding to Thyroxine

Abstract A DNA aptamer that selectively binds to a hormone, thyroxine, was isolated using the in vitro selection method. A pool of single-stranded DNAs consisting of random sequences was incubated with the thyroxine-immobilized agarose gel; bound DNAs were then collected and amplified by the polymerase chain reaction. This selection process was repeated nine times. The selected DNAs were cloned and sequenced. Some of the cloned DNA contained a consensus sequence. Several DNAs containing the sequence were chemically synthesized and the binding ratios to the thyroxine-immobilized gel were determined. Among them a DNA containing 14 nucleotides was found to bind selectively to thyroxine.

Isolation of DNAs that selectively bind a baculovirus produced mutant p53 (Ala 143) protein but not an RRL or WGL produced mutant p53 protein

A PCR-based technique was used to generate a large pool of random sequence double-stranded DNAs. Four DNA sequences that selectively bound in vitro to a mutant p53 143A protein, synthesized in baculovirus infected cells, were characterized. The four DNA sequences all approximated the known consensus sequence for wild-type p53. Wild-type p53 also bound the four DNA sequences. Two other mutant p53 proteins (His 175 or Trp 248) did not bind. The ability of mutant p53 143A protein to bind to DNA is totally dependent on the irt vitro system used to synthesize the p53 protein. Rabbit reticulocyte lysate (RRL) or wheat germ lysate (WGL) produced mutant p53 143A is unable to bind to DNA but does bind to a known protein partner, hdm2, thus these activities can be uncoupled.

In Vitro Selection of a DNA Ligand that Selectively Binds to the Anticancer Agent Methotrexate

Abstract A DNA ligand that selectively binds to the anticancer agent methotrexate was identified using the in vitro selection method. A pool of single-stranded DNAs consisting of random sequences was incubated with a methotrexate-immobilized agarose gel; bound DNAs were then collected and amplified using the polymerase chain reaction (PCR). This selection process was repeated six times. The selected DNAs were cloned and were found to contain two common sequences. One of these selected DNA clones selectively bound to the methotrexate-immobilized gel and showed a high level of discrimination against the gel immobilized with folic acid, which has a similar chemical structure to methotrexate. In the selection process, a buffer solution was used for DNA binding to the methotrexate-immobilized gel and water was used for elution from the gel. Ultraviolet absorption measurement showed that the conformation of the DNA ligand in the binding buffer was different from that in elution water.

Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures.

We have isolated a set of ligand-binding DNA sequences from a large pool of random sequence DNAs by selection and amplification in vitro, using similar methods to those described for the isolation of ligand-binding RNAs. The ligand-DNA interactions are both sequence- and ligand-specific, and are dependent on proper folding of the single-stranded DNA. Some ligands led to the isolation of more DNA sequences than RNA sequences, and vice versa. Analysis of individual sequences reveals that ligand binding is DNA-specific; RNAs of identical sequence could not interact with the same ligands. Ligand-binding DNAs might be more suitable than RNAs as potential pharmacological reagents because of the greater stability of DNA. The apparent primacy of RNA in the early evolution of life may have been due to its availability rather than to its functional superiority.