Sequence-directed curvature of DNA

DNAs from both prokaryotic and eukaryotic organisms have yielded restriction fragments which manifest markedly anomalous electrophoretic behaviour (reduced mobility) when run on polyacrylamide gels. We have shown previously that the abnormal electrophoretic behaviour of one such fragment is a consequence of stable curvature of the helix axis in solution. The molecules involved tend to contain oligo(dA)-oligo(dT) runs which are approximately in-phase with the helix repeat; however, the precise structural elements responsible for DNA curvature have not been identified. One popular model for curvature invokes a non-coplanar 'wedge-like' conformation of ApA/TpT dinucleotide pairs. Despite a lack of direct evidence in support of this model, it has been used to provide quantitative estimates of curvature. To critically evaluate the ApA wedge model, we have performed an electrophoretic analysis of a series of closely related DNA polymers in which oligo(dA)-oligo(dT) runs of different polarity were compared. We conclude that ApA dinucleotide wedges cannot account for DNA curvature. Therefore, quantitative estimates for ApA wedge deformations, based solely on apparent curvature, cannot be correct.     

Re-routing of a secretory protein by fusion with human growth hormone sequences

Cells with electron-dense secretory vesicles use them to store only specialized secretory products such as peptide hormones; other types of secreted proteins are externalized by an alternative, constitutive route. One possible mechanism for such segregation is that proteins destined for dense secretory vesicles contain unique 'sorting domains' that allow for selective targeting. Here, we set out to determine whether a constitutively secreted protein could be diverted to the dense secretory vesicles by attachment to a peptide hormone sequence. We made use of the ability of the mouse pituitary tumour cell, AtT-20, to correctly sort exogenous secretory proteins introduced into them by DNA transfection. We constructed a plasmid encoding a hybrid protein in which a constitutively secreted viral protein was fused to the carboxy terminus of human growth hormone (hGH). Cells expressing the hybrid protein were found to target it to dense secretory vesicles with an efficiency close to that observed for the parental hGH. These results support the hypothesis that sorting domains on peptide hormones direct their packaging into dense secretory vesicles. The results also suggest that proteins secreted by the constitutive pathway either do not contain any sorting domain, or their sorting signals can be overridden by those which direct peptide hormones.     

Structure of pre-pro-von Willebrand factor and its expression in heterologous cells

Von Willebrand factor (vWF), a multifunctional haemostatic glycoprotein derived from endothelial cells and megakaryocytes, mediates platelet adhesion to injured subendothelium and binds coagulation factor VIII in the circulation. Native vWF is a disulphide-bonded homopolymer; the monomeric subunits, of apparent relative molecular mass (Mr) 220,000 (220K) are derived from an intracellular precursor estimated at 260-275K. Multimer assembly is preceded by the formation of dimers, linked near their C-termini, which then assemble into filamentous polymers. The importance of the removal of the large vWF pro-polypeptide during multimer assembly, and whether this or other stages of the complex post-translational processing require components specific to endothelial cells or megakaryocytes, is unknown. Here we report an analysis of the complete sequence of pre-pro-vWF and expression of the molecule in heterologous cells. The vWF precursor is composed of several repeated subdomains. When expressed in COS and CHO cells, it is cleaved and assembled into biologically active high relative molecular mass disulphide bonded multimers. This suggests that the information for assembly of this complex molecule resides largely within its primary structure.     

Long-range colour-generating interactions across the retina

The existence of colour-generating interactions across the corpus callosum has recently been suggested from observations with a 'split-brain' patient, thus indicating long-range colour computations at the cortical level. Observations on induced colours described here suggest long-range colour computations at the retinal level. If a white surface surrounded by a particular colour is fixated for some time, the resulting after-image has two colours: the surround appears in complementary colour, whereas the white centre takes on the colour of the surround. The question of whether such colour induction is located in the retina or more centrally was tested in a brain-injured patient with hemianopia. It could be demonstrated that areas of the visual field that are no longer represented in the geniculo-striatal pathway still contribute to colour induction, suggesting that colour induction is a retinal phenomenon.     

Ratio between large and small carboxy-terminal molecular forms of cholecystokinin in brains of different species

The ratio between large and small carboxy-terminal forms of cholecystokinin in brain extracts from man, pig, dog, rat, chicken, frog and trout was determined by two sequence-specific radioimmunoassays. It was found that the relative amounts of large forms of cholecystokinin; are higher in mammalian brain than in brains of lower species.     

Immunoquantitation of some cytochrome P-450 isozymes in liver microsomes from streptozotocin-diabetic rats

Streptozotocin-diabetes in rats leads to a decrease of cytochrome P-450 UT-A (the major form in control rats) and an increase of cytochrome P-450 PB-B (the major one induced by phenobarbital treatment) in liver microsomes. The increased benzphetamine-N-demethylase activity can be related to the induction of cytochrome P-450 PB-B.