Using a CCD camera imaging system as a recording device to quantify human DNA by slot blot hybridization.

Slot blot hybridization of membrane-immobilized, single-stranded human DNA with the higher primate-specific alphoid probe D17Z1 is routinely used in forensic science to estimate the amount of DNA in biological samples. Typically, a chemiluminescent signal captured on film records the hybridization, and the quantity of the signal is related to the amount of immobilized DNA. Digital imaging using a cooled CCD camera offers an alternate non-film-based method for image acquisition with comparable sensitivity of detection, a greater dynamic range, enhanced capability of data interpretation, and often faster results than film. In addition, the data support the premise that more accurate and precise human DNA quantification should be obtained by not assuming a linear response of signal to known standards. Instead, quantity should be estimated using a second-order standard curve (R2 = 0.999). Finally, a CCD camera imaging system offers versatility for image capture of different signal sources and analysis of samples on a variety of support media.

Inhibition of antagonist and agonist binding to the human brain muscarinic receptor by arachidonic acid.

Arachidonic acid (AA) inhibits the binding of [3H]quinclidinyl benzilate ([3H]QNB) to the human brain muscarinic cholinergic receptor (mAChR). AA inhibits at lower concentrations in the absence of glutathione (I50 = 15 microM) than in the presence of glutathione (I50 = 42 microM). Inhibition of mAChR binding shows specificity for AA and is reduced with loss of one or more double bonds or with either a decrease or increase in the length of the fatty acid chain. Metabolism of AA by the lipoxygenase, epoxygenase, or fatty acid cyclooxygenase pathways is not required for the inhibitory activity of AA on mAChR binding. Inhibition of [3H]QNB binding by AA is reversible. While decreasing Bmax, AA increased the apparent KD for [3H]QNB and for the more polar antagonist [3H]NMS. In addition, AA inhibits binding of the agonist [3H]oxotremorine-M (I50 = 60 microM) and is the first mediator of mAChR action to be shown to reversibly inhibit mAChR binding. The feedback inhibition of the mAChR by AA may serve a homeostatic function similar to the reuptake and hydrolysis of acetylcholine following cholinergic nerve transmission.

Chemiluminescent detection of DNA probes in forensic analysis.

The conditions for hybridization and detection of enzyme-labeled probes have been optimized in our laboratory for use with oligonucleotides coupled to alkaline phosphatase. We have examined several enzyme-linked probe which are complimentary to commonly used variable number of tandem repeats (VNTR) loci to determine the feasibility of using chemiluminescence for routine application in forensic DNA analysis. It was found that a chemiluminescent detection system employing an alkaline phosphatase activated dioxetane in the presence of chemiluminescent enhancers provides a high degree of sensitivity in hybridization protocols with a significant savings in overall filter processing time. The chemiluminescent system achieved equal or greater sensitivity that observed for 32P-labeled probes in much shorter development times. Furthermore, a new chemiluminescent substrate, Lumi-Phos Plus, has recently been investigated and found to further decrease the filter development time for forensic assays.

Simple protocols for typing forensic biological evidence: chemiluminescent detection for human DNA quantitation and restriction fragment length polymorphism (RFLP) analyses and manual typing of polymerase chain reaction (PCR) amplified polymorphisms.

Methods for identity testing are described that enable extraction of DNA from biological samples, determination of the quantity of human DNA, and genetic analyses of the materials using restriction fragment length polymorphism (RFLP) typing and/or amplified fragment length polymorphism (AMP-FLP) typing of PCR products. The salient features of the procedures are simplicity, manual typing, nonradioactive chemiluminescent assays or silver staining for detection, and low cost. Most application-oriented laboratories involved in forensic and/or paternity testing should be able to implement these procedures.

Rapid isolation of DNA from complex biological samples using a novel capture reagent--methidium-spermine-sepharose.

We have synthesized and analyzed the functional properties of a novel DNA capture reagent containing a methidium moiety attached to a sepharose bead by a spermine linker. DNA present in a biological fluid or other complex sample binds to the reagent. The DNA-capture reagent complex is then separated from the sample by centrifugation and the DNA is released from the reagent by brief incubation in 0.1 to 0.5 N NaOH or KOH. Capture of DNA from complex samples is independent of the salt concentration of the sample, and occurs in the presence of high concentrations of EDTA, proteinase K and detergents. Many samples can be processed simultaneously. The following specific applications, in which denatured DNA is quantitated or characterized, are demonstrated: 1). Isolation of hepatitis B virus DNA from serum and quantitation by dot-blot hybridization, 2). Isolation and quantitation of DNA from urine, 3). Isolation of human genomic DNA from one microliter of blood or 100 HeLa cells followed by amplification of a specific gene sequence using the Polymerase Chain Reaction, 4). Isolation of single stranded phage M13 sequencing templates from bacterial cultures. These investigations suggest that a capture reagent containing an intercalating moiety bound to a solid support may be useful for many applications in molecular biology and molecular diagnostics.

Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA.

A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine and the 4-position of cytosine. These nucleotides are incorporated into DNA probes by standard nick-translation protocols. DNA probes labeled with biotin derivatives of these nucleotides are effectively hybridized to target DNA sequences and can be detected by a streptavidin and calf intestinal alkaline phosphatase conjugate with a sensitivity (0.25 pg DNA) sufficient for reproducible and rapid detection of single copy genes in a Southern blot of mammalian DNA. Also, a procedure has been developed to allow reprobing of nylon filters that have been hybridized with biotinylated probes and developed with the streptavidin/alkaline phosphatase conjugate and a standard dye system.

Gyrase . DNA complexes visualized as looped structures by electron microscopy.

Gyrase bound to duplex DNA in the absence of ATP is seen by electron microscopy as a nearly spherical particle frequently located at the intersection of two duplex DNA strands. Such looped structures with gyrase situated at the base of the loops are observed with both linear and circular DNA substrates, and two or three individual DNA molecules bound to the same protein are also seen at high DNA concentrations. Addition of the nonhydrolyzable beta,gamma-imido analog of ATP to the gyrase . DNA reaction mixture prior to sample fixation for microscopy reduces the frequency of gyrase molecules found at DNA intersections. Looped structures similar to those of the gyrase . DNA complex are also seen with the complex of DNA and the A subunit of gyrase. When negatively supercoiled DNA which has been partially relaxed by gyrase in the absence of ATP is fixed for electron microscopic examination, intermediate forms are observed that contain both supercoiled and relaxed loops in a single DNA molecule, with the enzyme located at the common base of the loops. These results suggest that gyrase possesses multiple DNA-binding sites, a feature which allows the enzyme to hold DNA in constrained loops. The relation of these observations to the mechanism of gyrase action is discussed.

Stabilization of Z-DNA by polyarginine near physiological ionic strength.

The identification of left handed or Z-DNA in solutions of poly d(GC) in high salt suggests that left handed DNA may exist in biological systems if stabilized at lower ionic strength. In the present study we show that binding of polyarginine to the Z form of poly d(GC) results in a protein-Z-DNA complex stable near physiological ionic strength. The percentage of Z-DNA in the low salt polyarginine-poly d(GC) complex was measured from the DNA circular dichroism spectrum. The ratio of Z to B-DNA is a linear function of polyarginine concentration and is sensitive to proteolytic digestion by trypsin. These results suggest that arginine-rich proteins may stabilize Z-DNA in vivo.

Deoxyribonucleic acid gyrase-deoxyribonucleic acid complex containing 140 base pairs of deoxyribonucleic acid and an alpha 2 beta 2 protein core.

Staphylococcal nuclease digestion of the complex between DNA and DNA gyrase yields a gyrase-DNA core particle composed of a 140 base pair DNA segment and an active gyrase enzyme. The partial specific volume and S20,w of this purified core complex are measured to be 0.70 cm3/g and 14.5 S, respectively, by sedimentation measurements in H2O and D2O media. The molecular weight of the core complex estimated from equilibrium centrifugation is 470 000; the ratio of the translational frictional coefficient to that of the unsolvated equivalent sphere is calculated to be 1.9. Treatment of free gyrase in solution with dimethyl suberimidate gives three cross-linked species of roughly equal amounts that can be identified as alpha 2, alpha 2 beta, and alpha 2 beta 2. When the gyrase core complex is treated with the same cross-linking agent, 70-80% of the protein is converted to the alpha 2 beta 2 species. These results establish that the gyrase-DNA core complex contains a 140 base pair DNA segment and a tetrameric alpha 2 beta 2 protein.

31P NMR studies of the solution structure and dynamics of nucleosomes and DNA.

31P NMR studies of 140 base pair DNA fragments in nucleosomes and free in solution show no detectable change in the internucleotide 31P chemical shift or linewidth when DNA is packaged into nucleosomes. Measurements of 31P spin-lattice relaxation times T1 and 31P-[H] nuclear Overhauser enhancements revealed internal motion with a correlation time of about 4 x 10(-10) sec in double helical DNA, both free in solution and bound to nucleosomal core proteins. This result implies greater dynamic mobility in double helical DNA than has previously been supposed.

Transient electric dichroism studies of nucleosomal particles.

We report transient electric dichroism experiments on nucleosomal core particles containing 140 and 175 base pairs of DNA, and on spacerless dinucleosomes. The results indicate that all particles posses a permanent dipole moment. The orientation time of 140 base pair nucleosomes implies an estimated maximum dimension of a = 130 A (a must be at least 111 A), consistent with the disk model. The maximum dimension of the spacerless dinucleosome is estimated to be about 290 A (at least 180 A), ruling out a structure in which two disks are stacked directly on top of each other. The reduced dichroism amplitude indicate that the DNA superhelix axis in nucleosomes aligns perpendicular to the electric field, as expected for a dipole moment directed along a C2 symmetry axis across the disk diameter. Nucleosomes containing 175 base pairs of DNA show a substantially larger dichroism amplitude that do 140 base pair nucleosomes. In the context of the disk model, this result is shown to be consistent with 100 base pairs of DNA per superhelical turn, but not with 80 base pairs per turn.

Physical studies of nucleosome assemble.

Biochemical, spectroscopic, and hydrodynamic studies were performed on the reconstituted complex of 140 base pair DNA and the arginine-rich histone tetramer (H3/H4)2. The histones bind to DNA in a 1:1 molar ration to form a stable particle which orients in an electric field with a rotational correlation time of 6.3 mus and a limitign reduced dichroism of --0.74. The complex was modeled hydrodynamically as a cylinder of dimensions 450 X 80 X 80 A containing approximately 1.5 superhelical turns. Addition of the lysinerich histones to this complex cause a condensation of the structure and results in physical properties nearly identical with those of a native nucleosomal particle.

Isolation and characterization of a spacerless dinucleosome from H1-deleted chromatin.

Calf thymus chromatin, depleted in histone H1, was digested with micrococcal nuclease and fractionated by column chromatography. 140 base pair nucleosome core particles were isolated along with an unusual particle containing 2 histone octamers and 240 base pairs of DNA. Evidence is presented that the spacer DNA region is absent from these modified dinucleosomes, which appear as stable products of the digestion process. The physical properties of both particles are presented along with brief speculation on their possible origin and function.