A protein kinase isolated from porcine brain microvessels is similar to a class of heat-shock proteins.

To further characterize a protein kinase present in porcine brain microvessels, a cDNA library using porcine microvessel poly(A) RNA was screened with polyclonal antibodies raised against the native protein kinase. Since no full-length cDNA clone could be obtained, the missing sequence information was completed using two subsequent polymerase chain reactions. The amplified transcripts were cloned and the sequence determined. Additionally, a genomic DNA library from porcine kidney was screened to substantiate the results obtained from the polymerase chain reaction. Earlier hints of a relation to a subclass of the family of heat-shock proteins (HSPs) based upon a close sequence similarity at its amino-terminus could be confirmed by comparison of the full-length cDNA sequences. Common protein kinase consensus sequences, a targeting sequence for proteins of the endoplasmic reticulum at the carboxy-terminus as well as a hydrophobic leader sequence in the amino-terminal region of the protein could also be identified. Furthermore, a set of membrane-associated substrate proteins of this enzyme could be detected in brain capillaries. The results indicate that at least some members of the HSP 90 subfamily undergo autophosphorylation and show protein kinase activity by phosphorylating substrate proteins in vitro.

DNA engineering shows that nucleosome phasing on the African green monkey alpha-satellite is the result of multiple additive histone-DNA interactions.

The mechanism underlying sequence-specific positioning of nucleosomes on DNA was investigated. African green monkey alpha-satellite DNA was reconstituted in vitro with histones. Histone octamers were found to adopt one major and several minor positions on the satellite repeat unit, very similar to those positions found previously in vitro, demonstrating that sequence-specific histone-DNA interactions are responsible for nucleosome positioning on this DNA. In order to understand the nature of these interactions in more detail, we have constructed a variant satellite fragment containing an insertion of half a helical DNA turn. The parent fragment directs histones to one major and two overlapping minor positions that are all affected by the insertion. All three frames respond in a unique fashion to the additional five base-pairs. From a quantitative analysis of the nucleosome positions on the engineered fragment, consensus "phasing boxes" as the basis for nucleosome positioning can be ruled out. Instead, our results argue very strongly that nucleosome positioning is due to the independent contribution of many different DNA-histone contacts along the entire core particle, in an apparently additive fashion.

Reconstitution of mononucleosomes: characterization of distinct particles that differ in the position of the histone core.

Reconstitution of mononucleosomes from DNA and core histones was carried out to study the positioning of histone octamers on the DNA. Using random DNA molecules in the 200 to 250 bp size range we found that the reconstitution products consisted of a mixture of three different types of particles that could be separated by low ionic strength gel electrophoresis. In one particle, DNA was complexed with histones along its entire length indicating the binding of more than one histone octamer. The second particle contained only one histone core that was always associated, however, with the terminal 145 bp of the DNA regardless of its sequence which can be ascribed to a DNA end effect. Only the third particle consisted of histone octamers bound at internal positions of the DNA and is therefore the only particle suitable for investigating the influence of the DNA sequence on the positioning of the histone cores. A defined 154 bp pBR 322 restriction fragment that contains three BspRI restriction sites was also reconstituted with core histones. The accessibility of these sites to BspRI was measured in order to delineate the utility of restriction nucleases as probes for the structure of chromatin. Two sites located close to the center of the DNA were less susceptible by at least a factor of 1000 as compared to free DNA while the susceptibility of the third site in the terminal section of the DNA decreased about 50 fold after reconstitution.

Sequence specificity of exonuclease III from E. coli.

The influence of the nucleotide sequence on the digestion of deoxyribonuclease from E. coli, has been investigated. It was found that the rate at which mononucleotides are released varies in a sequence dependent fashion. C-residues are cleaved off rapidly and G-residues slowly while A and T are released at an intermediate rate. Quantitative analyses of digestion experiments with synthetic DNA fragments made it possible to determine rate constants for the cleavage of several dinucleotide bonds by exonuclease III. These values were found to differ by up to a factor of 3. Summation of the differences can lead to appreciable variation in the overall rate of digestion of a DNA strand. The nucleotide specificity of exonuclease III leads to a transient appearance of a series of discrete DNA fragments intermediate in digestion and a stable set of fragments in limit digests, i.e. at the point when all DNA has become single-stranded. This property of exonuclease III needs to be taken into account for the application of the enzyme in the analysis of nucleoprotein complexes.