IL-6 protein production by airway epithelial(-like) cells disabled in IL-6 mRNA degradation

IL-6 mRNA and protein expression in human airway epithelial-like H292 cells depends on rapid, but regulable IL-6 mRNA degradation. We restricted IL-6 mRNA degradation by partially inhibiting protein synthesis and studied the IL-6 response. Despite partial inhibition of protein synthesis, IL-6 protein production was increased and prolonged. Furthermore, the threshold concentration for stimuli of IL-6 protein production decreased and the dose-response curves became steeper. Similar findings were obtained with primary human bronchial epithelial cells. This exaggerated production may apply to other proteins encoded by labile mRNA and is likely to occur during viral infection of airway epithelial cells.     

Precise location of DNase I cutting sites in the nucleosome core determined by high resolution gel electrophoresis.

The precise locations of the DNase I cutting sites in the nucleosome core have been determined by analysis of the DNA products of a DNase I digestion of 32P end-labelled mucleosome cores on a high resolution gel electrophoresis system. This system is capable of resolving fragments of mixed sequence DNA differing by one base into the region of 160 bases in length. The DNase I cutting sites in the core are found to be spaced at multiples of about 10.4 (i.e. clearly different from 10.0) bases along the DNA, but show significant variations about this value. In addition to the location of the sites, the stagger between individual sites on opposite strands has been determined and is found to be inconsistent with at least one proposed mechanism for nuclease cleavage of chromatin DNA. Finally, a calculated distribution of fragment lengths in a DNase I digest of nuclei has been determined from the data obtained from the nucleosome core and found to be in reasonable agreement with the observed distribution. The periodicity of 10.4 is discussed with respect to the number of base pairs per turn of chromatin DNA and the number of superhelical turns of DNA per nucleosome.     

DNase II digestion of the nucleosome core: precise locations and relative exposures of sites.

The precise locations and relative exposures of the DNase II-accessible sites in the nucleosome core DNA are determined using techniques previously employed for the enzyme DNase I. It is found that there are a number of similarities between the site exposure patterns for the two enzymes but that in general the DNase II seems to discriminate less among adjacent sites' accessibilities than does DNase I. The two enzymes attack essentially the same positions in the DNA, the average difference between the precise location of the site being less than one-half base for the two enzymes. Such close similarities in the digestion patterns of two enzymes with such different mechanisms of scission show that the patterns reflect the structure of the nucleosome core and not merely the properties of the particular enzyme used.     

The helical periodicity of DNA on the nucleosome

The precise number of base pairs per turn of the DNA double helix in the nucleosome core particle has been the subject of controversy. In this paper the positions of nuclease cutting sites are analysed in three dimensions. Using this midpoint of the DNA on the nucleosome dyad as origin, the cutting site locations measured along a strand of DNA are mapped onto models of the nucleosome core containing DNA of different helical periodicities. It is found that a helical periodicity of 10.5 base pairs per turn leads to cutting site positions which are sterically inaccessible. In contrast, a periodicity of 10.0 base pairs per turn leads to cutting site positions which are not only sterically sound, but which fall into a pattern such as would be expected when the access of the nuclease to the DNA is restricted by the presence of the histone core on one side and of the adjacent superhelical turn of DNA on the other. As proposed earlier by us (1), a value for the helical periodicity close to 10 base pairs per turn on the nucleosome, taken together with a periodicity close to 10.5 for DNA in solution - a value now established - resolves the so-called linkage number paradox.     

Structural aspects of proton-pumping ATPases

ATP synthase is found in bacteria, chloroplasts and mitochondria. The simplest known example of such an enzyme is that in the eubacterium Escherichia coli; it is a membrane-bound assembly of eight different polypeptides assembled with a stoichiometry of alpha 3 beta 3 gamma 1 delta 1 epsilon 1 a1b2c10-12. The first five of these constitute a globular structure, F1-ATPase, which is bound to an intrinsic membrane domain, F0, an assembly of the three remaining subunits. ATP synthases driven by photosynthesis are slightly more complex. In chloroplasts, and probably in photosynthetic bacteria, they have nine subunits, all homologues of the components of the E. coli enzyme; the additional subunit is a duplicated and diverged relation of subunit b. The mammalian mitochondrial enzyme is more complex. It contains 14 different polypeptides, of which 13 have been characterized. Two membrane components, a (or ATPase-6) and A6L, are encoded in the mitochondrial genome in overlapping genes and the remaining subunits are nuclear gene products that are translated on cytoplasmic ribosomes and then imported into the organelle. The sequence of the proteins of ATP-synthase have provided information about amino acids that are important for its function. For example, amino acids contributing to nucleotide binding sites have been identified. Also, they provide the basis of models of secondary structure of membrane components that constitute the transmembrane proton channel. An understanding of the coupling of the transmembrane potential gradient for protons, delta mu H+, to ATP synthesis will probably require the determination of the structure of the entire membrane bound complex. Crystals have been obtained of the globular domain, F1-ATPase. They diffract to a resolution of 3-4 A and data collection is in progress. As a preliminary step towards crystallization of the entire complex, we have purified it from bovine mitochondria and reconstituted it into phospholipid vesicles.     

Topological measurement of an A-tract bend angle: variation of duplex winding

The rotational variant method of Lutter et al. was developed to measure the bend angle induced when a protein binds to DNA. To measure the intrinsic bend conferred by a sequence of six adenine bases (an A6 tract), the method was modified by relaxing at high temperature to remove the bend. We describe here an alternative approach that involves unwinding the duplex DNA between adjacent bends in plasmids containing tandemly repeated blocks of A-tracts. This method measures the topological difference contributed by adjacent bends when they are in two different rotational settings, and therefore does not require reference to a straight state. The interbend DNA was unwound by use of the intercalator chloroquine, or, alternatively, by raising the temperature in the relaxation reaction. The effect of this unwinding is to change the pitch of the superhelix of the tandem repeats from which the bend angle is measured. The result is a bend angle value that is consistent with that measured using the bend-straightening version of the method. This version offers several advantages that complement the conventional bent versus straight approach.     

Topological measurement of an A-tract bend angle: effect of magnesium

Sequences of four to six adenine residues, termed A-tracts, have been shown to produce curvature in the DNA double helix. A-tracts have been used extensively as reference standards to quantify bending induced by other sequences as well as by DNA binding proteins when they bind to their sites. However, the ability of an A-tract to serve as such a standard is hampered by the wide variation of values reported for the amount of bend conferred by an A-tract. One experimental condition that differs in these studies is the presence of divalent cation. To evaluate this effect, a new application of a topological method, termed rotational variant analysis, is used here to measure for the first time the effect of the presence of magnesium ion on the bend angle conferred by an A-tract. This method, which has the unique ability to measure a bend angle in the presence or absence of magnesium ion, demonstrates that magnesium ion markedly increases the bend angle. For example, when measured in a commonly used gel electrophoretic buffer, the bend angle conferred by a tract of six adenine residues increases from about 7 degrees in the absence of magnesium ion to 19 degrees in the presence of 3.9 mM magnesium ion. This quantitative demonstration of substantial magnesium ion dependence has several important implications. First, it explains discrepancies among bend values reported in various previous studies, particularly those employing gel electrophoretic versus other solution methods. In addition, these findings necessitate substantial revisions of the conclusions in a large number of studies that have used A-tract DNA as the bend angle reference standard in comparison measurements. Finally, any such future studies employing this comparison methodology will need to use the same sequence analyzed in the original measurements as well as replicate the original measurement conditions (e.g. ionic composition and temperature).