Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).     

The primase activity of DNA polymerase alpha from calf thymus

The nearly homogeneous 9 S DNA polymerase alpha from calf thymus contains a primase activity that allows priming of DNA synthesis on single-stranded templates in the presence of ribonucleoside triphosphates. Both on synthetic and natural single-stranded templates, RNA primers of 8-15 nucleotides in length are formed. In the absence of dNTPs, primers of some hundred nucleotides in length are observable. ATP and/or GTP are required for the priming reaction. UTP and CTP cannot initiate the RNA synthesis. M13 single-stranded DNA can be converted to the nicked double helical form upon primase-primed replication by the 9 S enzyme. Priming occurs mostly at specific sites on the M13 genome and replication products of up to 6000 nucleotides in length are formed. In the presence of the single-stranded DNA binding protein from Escherichia coli, specificity of priming is strongly increased. The primase is inhibited by salt and actinomycin; it is insensitive to alpha-amanitin and N-ethylmaleimide. Due to the strong complex formation between DNA polymerase and primase, it has not been possible to separate the two activities of the multisubunit 9 S enzyme.     

Complexation and phase transfer of nucleotides by gramicidin S

Gramicidin S (GrS), an amphiphilic cyclosymmetric decapeptide produced by Bacillus brevis G-B and Nagano, binds nucleotides in water to yield a complex which partitions into organic solvents. The observed phase-transfer efficiencies at a given pH increase in the order AMP less than ADP less than ATP. The lipophilic complexes have well-defined stoichiometries, which were determined to be 1:1 for ADP-GrS at pH 7 and ATP-GrS at pH 3 and 1:2 for ATP-GrS at pH 7. The interaction is primarily ionic, involving coordination of the ornithine N delta H3+ groups of the peptide and the phosphoryl groups of the nucleotide, with little contribution from the nucleoside moiety. Exchange of organic and inorganic phosphates was also found to be mediated by GrS. The nucleotide complexes are sparingly soluble in water and self-associate extensively in CHCl3, most likely by cross-beta-aggregation, to yield large, ribbonlike aggregates which give rise to broad NMR resonances. Structures for the 1:1 and 1:2 complexes are proposed. In the latter, two GrS molecules envelop the nucleotide, orienting their apolar faces externally in opposite directions, while the lateral faces retain considerable polar character and direct aggregation in organic media. The 1:1 complex possesses a single apolar face and is less lipophilic. Binding constants were estimated by simulation of the extraction data. For the 1:1 complexes, K1:1 congruent to 4 X 10(4) M-1 for either ADP or ATP. Phase transfer of the ATP complex at pH 7 could be modeled either by stochastically independent binding to two noninteracting sites on the nucleotide with K1 approximately K2 approximately K1:1 or by a sequential process with K1 approximately K1:1 and K2/K1 less than 100. It is concluded that the apparent selectivity of GrS for ATP over ADP is a consequence of the greater lipophilicity and tendency to aggregate of the 1:2 complex, rather than an intrinsically higher binding affinity for triphosphates. GrS is, to our knowledge, the first peptide known to possess phase-transfer activity toward nucleotides; this is, in addition, the first molecular recognition process in which GrS is demonstrated to participate in vitro at physiologically active concentrations.     

Ras induces anchorage-independent growth by subverting multiple adhesion-regulated cell cycle events.

Anchorage-independent growth is a hallmark of transformed cells, but little is known of the molecular mechanisms that underlie this phenomenon. We describe here studies of cell cycle control of anchorage-independent growth induced by the ras oncogene, with the use of a somatic cell mutant fibroblast line (ER-1-2) that is specifically defective in oncogene-mediated, anchorage-independent growth. Control, nontransformed PKC3-F4 cells and ER-1-2 cells cannot proliferate in semisolid medium. Three important cell cycle events are dependent on adhesion of these cells to a substratum: phosphorylation of the retinoblastoma protein, pRB; cyclin E-dependent kinase activity; and cyclin A expression. PKC3-F4 cells that express ras (PKC3-F4/ras cells) proliferate in nonadherent cultures, and each of these three events occurs in the absence of adhesion in PKC3-F4/ras cells. Thus, ras can override the adhesion requirement of cellular functions that are necessary for cell cycle progression. ER-1-2 cells that express ras (ER-1-2/ras cells) possess hyperphosphorylated forms of pRB and cyclin E-dependent kinase activity in the absence of adhesion but remain adhesion dependent for expression of cyclin A. The adhesion dependence of pRB phosphorylation and cyclin E-dependent kinase activity is therefore dissociable from the adhesion dependence of cyclin A expression. Furthermore, ectopic expression of cyclin A is sufficient to rescue anchorage-independent growth of ER-1-2/ras cells but does not induce anchorage-independent growth of PKC3-F4 or ER-1-2 cells. However, like pRB phosphorylation and cyclin E-dependent kinase activity, the kinase activity associated with ectopically expressed cyclin A is dependent on cell adhesion, and this dependence is overcome by ras. Thus, the induction of anchorage-independent growth by ras may involve multiple signals that lead to both expression of cyclin A and activation of G1 cyclin-dependent kinase activities in the absence of cell adhesion.     

Preferential outcrossing in the complex species Persoonia mollis R. Br. (Proteaceae)

The breeding system of the extremely diverse species Persoonia mollis (Proteaceae) was characterized to, firstly, assess its importance as a mechanism promoting diversity and, secondly, to investigate the mode of control over selective fruit abortion. Fruit quantity and quality was assessed following self-and outcross-pollination manipulations. Twenty percent of outcrossed flowers set fruit, compared to only 1% of flowers fertilized with self-pollen. Fruits produced by self-fertilization were 72% of the weight of cross-fertilized fruits. Fruits produced by self-fertilization were significantly fewer in number and lighter than fruits following natural pollination of unmanipulated flowers (17% fruit set), but outcrossed and naturally pollinated fruits were equivalent. Flower to fruit demography suggested that a post-zygotic mechanism may be preferentially selecting the most vigorous zygote genotypes, as ovary abscission occurs mostly between 4 and 30 weeks after pollination, regardless of pollen source. Self-pollen tube growth was found to be inhibited within the styly, while pollen tubes were found in the ovary for 50% of all outcrossed flowers. These data suggest that a pre-zygotic pseudo self-incompatibility mechanism is the cause of low fruit set following self-pollination. The breeding system of P. mollis was found to promote outbreeding, with an emphasis on flexibility and post-zygotic choice following pre-zygotic pseudo self-incompatibility.Publication no. 120 from the Ecology and Genetics Group of the University of Wollongong     

Colestipol-induced changes in LDL composition and metabolism. II. Studies in humans

We investigated the effect of the bile acid sequestrant, colestipol hydrochloride, on the composition and metabolism of human low density lipoprotein (LDL). Colestipol treatment produced a disproportionate decrease in LDL cholesterol compared to LDL apoB, resulting in a significant decrease in the LDL cholesterol/apoB ratio. Electron microscopy revealed that LDL particles were smaller in size and analytical ultracentrifugation demonstrated that colestipol therapy selectively depleted larger, more buoyant LDL particles of Sf degrees 6-7. Thus, colestipol therapy produced LDL that were smaller in size, more dense, and characterized by a decreased cholesterol to protein ratio. To determine whether the altered LDL had different metabolic properties, autologous LDL was isolated from subjects before and during colestipol therapy and their fractional catabolic rates (FCR) were then simultaneously determined in the same patient while on therapy. Eight LDL turnover studies comparing the catabolism of LDL isolated during therapy (Rx-LDL) and LDL isolated off therapy (Con-LDL) were performed in six subjects. All subjects responded to colestipol treatment, with an average 29% fall in LDL cholesterol. In four of six subjects, and in six of eight studies, the FCR of Rx-LDL was substantially slower than that of Con-LDL. These studies demonstrate that a drug intervention may alter subpopulations of LDL particles in such a way that overall LDL composition is changed. This alteration may independently affect the intrinsic metabolic behavior of the LDL. We suggest that such drug- (or dietary-) induced changes in LDL composition need to be considered in kinetic studies designed to assess the overall impact of the perturbation being studied.     

Genetic and Cytogenetic Analysis of the ADH Region in DROSOPHILA MELANOGASTER

Eighteen Adh-negative mutations were selected with 1-pentyn-3-ol after feeding of formaldehyde. Twelve of the 18 were shown by cytological and genetic analysis to be deletions. Cytological examination of the deletions allowed us to localize the Adh gene to a region including bands 35B3-5 on the left arm of chromosome 2. The deletions were also used to order known visible loci located near Adh.--The vital loci near Adh were also investigated. A total of 109 lethal mutations were generated with EMS and 33 of these, localized within a region defined by the overlap of two of the deletions, were found to belong to 13 complementation groups. If one includes three other loci known to belong there (el, Adh and Sco) a total of 16 complemetation groups have been identified in the region close to Adh.     

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.