Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions

The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl2 and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or 113Cd(II). The Co(II)2 NC protein shows d-d electronic transitions at 695 nm [epsilon = 675 M-1 cm-1 per Co(II)] and 640 nm [epsilon = 825 M-1 cm-1 per Co(II)] compatible with regular tetrahedral geometry around both Co(II) ions. The Co(II)2 and Cd(II)2 NC proteins show intense charge-transfer bands in the near-UV, at 355 nm (epsilon = approximately 4000 M-1 cm-1) and 310 nm (epsilon = approximately 8000 M-1 cm-1) for the Co(II) protein and 255 nm (epsilon = approximately 10(4) M-1 cm-1) for the Cd(II)2 NC protein, compatible with -S- coordination. 113Cd NMR of the 113Cd(II)2 NC protein shows two 113Cd NMR signals at 659 and 640 ppm, respectively, each integrating to approximately 1 Cd(II) ion. The downfield chemical shifts suggest coordination of each 113Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X2-Cys-X4-His-X4-Cys- sequence contained in the N-terminal half of the molecule. 113Cd NMR shows, however, that the sites are not identical. Isolation of the NC protein under standard aerobic conditions results in oxidation of the sulfhydryl groups and loss of the coordinated Zn(II) ions, while preparation of the NC protein as the Cd(II) derivative at low pH protects the sulfhydryl groups from oxidation.     

DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum(II)

The DNA unwinding produced by specific adducts of the antitumor drug cis-diamminedichloroplatinum(II) has been quantitatively determined. Synthetic DNA duplex oligonucleotides of varying lengths with two base pair cohesive ends were synthesized and characterized that contained site-specific intrastrand N7-purine/N7-purine cross-links. Included are cis-[Pt(NH3)2[d(GpG)]], cis-[Pt(NH3)2(d(ApG)]], and cis-[Pt(NH3)2[d(GpTpG)]] adducts, respectively referred to as cis-GG, cis-AG, and cis-GTG. Local DNA distortions at the site of platination were amplified by polymerization of these monomers and quantitatively evaluated by using polyacrylamide gel electrophoresis. The extent of DNA unwinding was determined by systematically varying the interplatinum distance, or phasing, in polymers containing the adducts. The multimer that migrates most slowly gives the optimal phasing for cooperative bending, from which the degree of unwinding can be obtained. We find that the cis-GG and cis-AG adducts both unwind DNA by 13 degrees, while the cis-GTG adduct unwinds DNA by 23 degrees. In addition, experiments are presented that support previous studies revealing that a hinge joint forms at the sites of platination in DNA molecules containing trans-GTG adducts. On the basis of an analysis of the present and other published studies of site-specifically modified DNA, we propose that local duplex unwinding is a major determinant in the recognition of DNA damage by the Escherichia coli (A)BC excinuclease. In addition, local duplex unwinding of 13 degrees and bending by 35 degrees are shown to correlate well with the recognition of platinated DNA by a previously identified damage recognition protein (DRP) in human cells.     

Nematode Temperature Responses: a Niche Dimension In Populations of Bacterial-Feeding Nematodes

The optimum temperatures for population development were determined for six species of bacterial-feeding nematodes from among eight temperatures, ranging from 5 to 40 C. Four of the species are cohabiting species. The range of temperatures over which population development occurs (temperature niche breadth) is different for the cohabiting species. This difference may be a means of reducing competition between species, thus increasing temperatures over which habitats can be exploited.     

Electron microscopic localization of lactate dehydrogenase in osteoclasts of chick embryo tibia

Synopsis Lactate dehydrogenase (LDH) was localized in osteoclasts of fixed and unfixed 19-day chick embryo tibias using a copper ferrocyanide capture reaction and osmiophilic polymer generation. This study revealed that: (1) LDH activity in fixed, briefly rinsed osteoclasts was associated principally with limiting membranes of cytoplasmic vacuoles and vesicles and with the plasma membrane; (2) LDH activity in unfixed osteoclasts was associated only with mitochondria; and (3) some mitochondria were stained in fixed tissue given a long rinse. These results indicate that: cytoplasmic LDH diffused out of unfixed tissue; mitochondrial LDH was inactivated by formaldehyde in fixed tissue; and formaldehyde-inhibited mitochondrial LDH can be reactivated by a long rinse. Although the vesicles that stained for LDH activity were found in all parts of the cell, they were concentrated near the ruffled border, and there is evidence that they contained material from the bone surface. These results suggest that the LDH associated with cytoplasmic vesicles of the osteoclast may be important in processing of material resorbed from the bone surface and that osteoclastic mitochondria may utilize lactate from the bone fluid for energy production.     

The development and fine structure of ultimobranchial glands in larval anurans

A comparative optical microscopic and ultrastructural study on the ultimobranchial (UB) glands of three common species of Israeli anurans: Bufo viridis, Hyla arborea, and Rana ridibunda during metamorphosis is presented. The UB glands typically consist of a single follicle with a central lumen, though occasionally secondary follicles are present in Hyla and Rana. A single UB cell type is found which appears either in a very electron-dense "dark" form or as a less dense "light" form, though the ratio of dark: light cells from gland to gland at any one stage of metamorphic development is quite variable. By the end of metamorphosis in Bufo and Hyla all the UB cells are usually of the light variety, whereas in Rana the dark cells persist. The organelles of these secretory cells including secretory granules, granular endoplasmic reticulum, free ribosomes, tonofilaments, microtubules, Golgi bodies, and lipid droplets, their distribution, abundance, and possible functions in relation to metamorphosis are described. Apocrine secretion into the central lumen of the gland is also described and discussed.     

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.     

The effect of restricted feeding on the wheel-running activity rhythms of the predatory marsupial Dasyurus viverrinus

Summary The effects of restricted feeding schedules on the circadian rhythms of wheel-running of Dasyurus viverrinus were examined under a light/dark cycle and in constant darkness (experiment 1) and in constant light (experiment 2). The results of the 2 experiments showed that: (1) in contrast to the light/dark cycle, restricted feeding is only a weak zeitgeber for the wheel-running activity rhythms of D. viverrinus; (2) restricted feeding elicits meal anticipatory activity in D. viverrinus comparable to that elicited by restricted feeding in the rat; (3) transient cycles of the anticipatory activity free-run with a period different to that of the main component of activity for several cycles after the termination of restricted feeding; and (4) activity suggestive of beating between 2 oscillators occurs during restricted feeding and after the termination of restricted feeding. Taken together the latter 3 observations suggest that the activity rhythms of D. viverrinus are controlled by at least 2 separate circadian oscillators.