Purification of sequence-specific DNA-binding proteins by affinity chromatography.

The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. Preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support, is described, and an alternate protocol provides a method to couple DNA to commercially available CNBr-activated Sepharose. A method for purification of crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin is also provided. A detailed protocol for the actual affinity chromatography procedure is described and a support protocol allows the investigator to determine the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.

Purification of sequence-specific DNA-binding proteins by affinity chromatography.

Affinity chromatography is a very effective and straightforward means of purifying a protein based on its sequence-specific DNA-binding properties. The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. The first basic protocol describes preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support. An provides a method to couple DNA to commercially available CNBr-activated Sepharose, and a support protocol describes how to purify crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin. The second basic protocol outlines the affinity chromatography procedure. A second support protocol describes determination of the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.

TUNEL-positive ganglion cells in human primary open-angle glaucoma.

OBJECTIVE: To determine whether retinal ganglion cell death in primary open-angle glaucoma occurs by apoptosis. METHODS: Eighteen eyes of 17 subjects with documented primary open-angle glaucoma were compared with 21 control eyes that were group matched for age, race, and sex. Staging of glaucoma severity was performed by histologic optic nerve evaluation. Fixed, paraffin-embedded retinal sections were assayed by the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (UTP)-biotin nick end-labeling) method to detect the internucleosomal DNA fragmentation that is characteristic of apoptosis. RESULTS: A positive TUNEL reaction was observed among ganglion layer cells in 10 of 18 cases with glaucoma, compared with 1 of 11 control cases without confounding systemic disease (5 control eyes were excluded owing to artifactual staining and 4 eyes had confounding systemic disease). Sections containing more than 250,000 cells in the ganglion cell layer were examined in cases and controls. The frequency of TUNEL-positive cells in the ganglion cell layer in cases with glaucoma was 1.76 per 10,000, or 15.2 times greater than the control frequency from individuals without confounding disease (P < .001; 95% CI, 2.46-623). Eyes without glaucoma from subjects with diabetes and amyotrophic lateral sclerosis showed more positive cells than other controls. CONCLUSION: Apoptosis seems to be a mechanism of cell death in human eyes with primary open-angle glaucoma.

Quantitative studies of elastin in the optic nerve heads of persons with primary open-angle glaucoma.

PURPOSE: To compare quantitative and qualitative differences in elastin content in the optic nerve heads of glaucomatous and control human eyes. METHODS: Transmission electron microscopy and quantitative histomorphometry on ten control and ten glaucomatous eyes. RESULTS: Elastin fiber complexes in the control lamina cribrosa were smaller and more numerous than in the insertion zone of the sclera immediately surrounding the lamina. Although the density of elastin fibers in the normal lamina was twice that of the insertion zone (P = 0.004), the percent area of the connective tissue matrix occupied by elastin was the same for both zones (P > 0.4). There was no difference between control and glaucomatous eyes in the quantified parameters of elastin content or in the ultrastructure of elastin between control and glaucomatous eyes. CONCLUSIONS: The authors demonstrated for the first time that elastin in the normal lamina consists of fibers of smaller diameter than in the adjacent sclera, although the total amount of elastin is similar in both locations. This may provide maximum viscoelasticity within the limited connective tissue beam area of the lamina. Despite using a large number of specimens, the authors again found no differences between normal and glaucomatous eyes in the number or ultrastructural appearance of elastin fibers.

D2 dopamine receptor involvement in the rough-and-tumble play behavior of juvenile rats.

By using dorsal contacts and pinning to quantify play behavior in juvenile rats, it was found that the D2 agonist, quinpirole, reduced both measures of play at doses greater than 0.05 mg/kg. Eticlopride, a D2 antagonist, also reduced both measures of play and blocked the reduction caused by quinpirole. The effect of quinpirole on play was largely unaffected by concurrent administration of either a D1 agonist (SKF 38393) or a D1 antagonist (SCH 23390), suggesting that D1 and D2 receptors are functionally independent with respect to play behavior. Quinpirole also reduced overall activity, suggesting that the effects on play may not be selective to neural circuitry responsible for play behavior. Although low doses of quinpirole (0.001--0.03 mg/kg) had a tendency to increase pinning, this effect was not very robust. These data suggest that D2 dopamine receptors may not have a major role in the control of play behavior in juvenile rats.

Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis.

PURPOSE: To investigate whether retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. METHODS: Chronic elevated eye pressure was produced in 20 monkey eyes, and the optic nerve was transected unilaterally in the orbit of 10 monkeys and 14 rabbits. Sixteen monkey and 14 rabbit eyes were studied as normal controls. Analytic methods included light and electron microscopy, histochemistry for DNA fragmentation (TUNEL method), and DNA electrophoresis in agarose gels. RESULTS: Dying ganglion cells in the experimental retinas exhibited morphologic features of apoptosis, including chromatin condensation and formation of apoptotic bodies. Cells with a positive reaction for DNA fragmentation were observed in eyes subjected to axotomy and experimental glaucoma but were only rarely encountered in control eyes. No evidence of internucleosomal fragmentation was detected electrophoretically, possibly because of the small proportion of cells that were dying at any given time. CONCLUSION: Some retinal ganglion cells injured by glaucoma and by axotomy die by apoptosis.

Primary open-angle glaucoma is not associated with photoreceptor loss.

PURPOSE: To determine if photoreceptors die in primary open-angle glaucoma. METHODS: Retinas were examined in a masked fashion from nine standard locations of 14 eyes with documented open-angle glaucoma and from nine age-matched control eyes. The number and density of photoreceptors, as well as the area and height of the outer nuclear layer, were calculated with an automated image analysis system. The number of photoreceptors per 0.1 mm of retina was determined. RESULTS: No significant difference was seen between control and glaucomatous eyes in comparisons of photoreceptor density, outer nuclear layer height, or photoreceptors per 0.1 mm of retinal length in nine retinal zones. There was no detectable association between photoreceptor number and severity of glaucoma (defined as mild, moderate, or severe), visual field, and optic nerve fiber loss. In eyes in which damage predominated in the upper or lower visual field, no corresponding difference in photoreceptor number in upper compared to lower retinal zones was observed. CONCLUSION: Photoreceptors are not lost in substantial numbers in primary open-angle glaucoma.

Periodic binding of individual core histones to DNA: inadvertent purification of the core histone H2B as a putative enhancer-binding factor.

By using a DNase I footprinting assay, we have purified a factor by DNA affinity chromatography that binds to the minimal enhancer region of the Drosophila knirps gene and subsequently identified the protein as the core histone H2B. This inadvertent purification of a core histone as a putative sequence-specific DNA binding protein was due to a previously unknown property of H2B to interact with DNA in a periodic manner. Moreover, we found that each of the individual core histones, but not histone H1 or high mobility group protein 1, bound to the knirps enhancer to give a repetitive DNase I footprint pattern with a periodicity of about 10 base pairs, which is approximately one turn of the DNA helix. In addition, preparations containing the core histones H2A-H2B or H3-H4 yielded identical periodic DNase I footprint patterns on several different promoter and enhancer regions. These findings suggest that there are periodic, homotypic interactions between DNA-bound core histones that result from an alteration of the overall DNA structure such as the curvature rather than a specific sequence. We have also shown that histones H2A-H2B can repress initiation of transcription by RNA polymerase II. The phenomena described here may reflect histone-DNA interactions in non-nucleosomal stretches of chromatin and could be involved in some aspects of either rotational or translational positioning of nucleosomes. Furthermore, these findings indicate that a repeated 10 bp DNase I ladder, which has previously been considered to be a property of an intact nucleosome, can also be generated with subnucleosomal components. It will thus be necessary to reevaluate the criteria applied to the analysis of nucleosomes both in vivo and in vitro.

Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription.

To understand the principles of control and selectivity in gene expression, the biochemical mechanisms by which promoter- and enhancer-binding factors regulate transcription by RNA polymerase II were analyzed. A general observed repressor of transcription was purified and identified as histone H1. Since many aspects of H1 binding to naked DNA resemble its interaction with chromatin, purified H1 bound to naked DNA was used as a model for the repressed state of the DNA template. Three sequence-specific transcription factors, Sp1, GAL4-VP16, and GAGA factor, were shown to counteract H1-mediated repression (antirepression). In addition, Sp1 and GAL4-VP16, but not the GAGA factor, activated transcription in the absence of H1. Therefore, true activation and antirepression appear to be distinct activities of sequence-specific factors. Furthermore, transcription antirepression by GAL4-VP16 was sustained for several rounds of transcription. These findings, together with previous studies on H1, suggest that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.

Sequence-specific transcriptional antirepression of the Drosophila Krüppel gene by the GAGA factor.

We have analyzed the proximal promoter of the Drosophila Krüppel (Kr) gene. A 44-base pair fragment containing the RNA start sites contains significant promoter activity, and this minimal promoter is flanked both upstream and downstream by binding sites for the GAGA factor. The GAGA factor is the predominant sequence-specific DNA binding factor that interacts with the Kr promoter region, and the purified protein activates Kr transcription in vitro. However, strong transcriptional activation of Kr as well as of Ultrabithorax, another GAGA factor-responsive gene, requires the presence of a DNA binding transcriptional repressor. The GAGA factor is able to relieve this repression in a binding site-dependent manner, and, thus, these data suggest that the GAGA factor functions as an antirepressor, rather than an activator, of the Kr gene.