Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein

The GTP-binding p21 protein, encoded by the ras-oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate-binding loop regions, Tyr 4-Thr 20, herein designated as segment 1, and Ile 55-Met 67, herein designated, as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF-tu) (Val 12-Thr 28 corresponds to segment 1; His 78-Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9-Cys 25 corresponds to segment 1 and Tyr 95-Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF-tu and ADK are similar to one another and that the conformation of the segment 2 region of EF-tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF-tu is strikingly similar to that of the two segments in ADK. In the originally proposed X-ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.     

Regulation of D-xylose utilization by hexoses in pentose-fermenting yeasts

The aldopentose D-xylose is one of the most abundant sugars in plant biomass and its efficient microbial utilization is of fundamental importance in the overall bioconversion of lignocellulosic materials into liquid fuels and chemicals. The discovery of pentose-fermenting yeasts in the early 1980's led to world wide interest because of the perceived potential for improved D-xylose fermentation to enhance the prospect of biomass conversions. However, the utilization of D-xylose by pentose-fermenting yeasts can be adversely affected by the hexoses, mainly D-glucose and D-mannose, which are usually present in high amounts in lignocellulosic hydrolysates. Research in the past several years has uncovered some of the regulatory effects of D-glucose on D-xylose utilization. However, much remains unknown about the mechanisms responsible for these effects. This review summarizes the current state of knowledge on the induction, repression and inactivation of D-xylose utilization in pentose-fermenting yeasts.     

Viewing molecules with scanning tunneling microscopy and atomic force microscopy

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.     

Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

Summary An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H⁺-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na⁺-K⁺-ATPase (Mayahara et al. 1980) and gastric H⁺-K⁺-ATPase (Fujimoto et al. 1986). K⁺-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na⁺-K⁺-ATPase and Ca²⁺-ATPase. The K⁺-independent NPPase activity was diminished by the inhibitors of H⁺-ATPase such as N-ethylmaleimide (NEM) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H⁺-ATPase which plays a role in acidification.     

Non-everted oxygenated rat intestinal segments as a measure of neutral detergent fiber effects on iron absorption

Iron absorption in the presence of varying amounts and sizes of dietary fiber was measured. A method using non-everted rat intestinal segments perfused in oxygen was refined. Neutral detergent fiber (NDF), a component of dietary fiber, was extracted from cooked pinto bean (Phaseolus vulgaris). The NDF did not affect iron absorption in intestinal segments from iron replete rats. However, 4 and 6 mg of NDF/ml significantly decreased iron absorption in the intestinal segments from anemic rats. NDF with a smaller particle size of 0.125 mm increased iron absorption relative to that absorbed with 0.180 mm particles. Histological examination validated using non-everted intestinal segments perfused with oxygen as a method for studying dietary effects on iron absorption. Segments which are not everted are less prone to damage. Perfusion with oxygen maintained metabolic activity in the tissue during the experiment.     

Bacterial interactions with silver

Summary This review examines interactions between bacteria and the biologically non-essential metal, silver. Aspects of silver toxicity, tolerance and accumulation (possible binding and uptake as opposed to energy-dependent transport) in bacteria are discussed. In addition, plasmid biology is examined briefly since little information is available on the exact mechanism(s) of plasmid-endoced silver resistance in bacteria.     

Structure and expression of ubiquitin genes of Drosophila melanogaster.

We isolated and characterized two related ubiquitin genes from Drosophila melanogaster, polyubiquitin and UB3-D. The polyubiquitin gene contained 18 repeats of the 228-base-pair monomeric ubiquitin-encoding unit arranged in tandem. This gene was localized to a minor heat shock puff site, 63F, and it encoded a constitutively expressed 4.4-kilobase polyubiquitin-encoding mRNA, whose level was induced threefold by heat shock. To investigate the pattern of expression of the polyubiquitin gene in developing animals, a polyubiquitin-lacZ fusion gene was introduced into the Drosophila genome by germ line transformation. The fusion gene was expressed at high levels in a tissue-general manner at all life stages assayed. The ubiquitin-encoding gene, UB3-D, consisted of one ubiquitin-encoding unit directly fused, in frame, to a nonhomologous tail sequence. The amino acid sequence of the tail portion of the protein had 65% positional identity with that of yeast UBI3 protein, including a region that contained a potential nucleic acid-binding motif. The Drosophila UB3-D gene hybridized to a 0.9-kilobase mRNA that was constitutively expressed, and in contrast to the polyubiquitin gene, it was not inducible by heat shock.