Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe.

The three-dimensional folding of Xenopus oocyte 5S rRNA has been examined using the coordination complex Rh(phen)2phi3+ (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)2phi3+ binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the 5S rRNA. On the wild-type 5S rRNA, strong cleavage is found at residues U73, A74, A101, and U102 in the E loop and U80 and G81 in helix IV; additional sites are evident at A22 and A56 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full 5S RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)2phi3+ do, however, indicate that the apposition of several noncanonical bases as well as stem--loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured.(ABSTRACT TRUNCATED AT 250 WORDS)     

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.     

Structural basis for the red light induced repolarization of tip growth in caulonema cells ofCeratodon purpureus

Summary Two dynamic changes are associated with the phytochrome-regulated phototropic response in tip cells of the mossCeratodon purpureus: a tip-located gradient shift of chlortetracy-cline (CTC)-stained calcium and a structural reorganization of apical microfilaments (MFs). We examined the interdependence of these processes. Cells were treated with the antimicrotubule drug oryzalin, the antimicrofilament drug cytochalasin-D, and the calcium channel blocker nifedipine. respectively. The effects on phototropic growth, on the structural alignment of the cytoskeleton (microtubules, MTs; microfilaments) and on the distribution of CTC-stained calcium were studied under each of these conditions. In gravitropically growing tip cells the apical MFs form a cortical collar-like structure, consisting of actin bundles with a parallel axial alignment. These MFs point towards the presumptive growing point, a weakly stained region in the tip of the cell from which bundles are absent. MTs are present in the cortex and in the endoplasm of the tip, predominantly oriented longitudinally. The MTs converge within the central apex. The cells show a steep tip-to-base CTC-calcium gradient with its highest signal in the central apex. Destruction of MTs by 1 M oryzalin induces several translocational effects: (i) the growing zone and phototropic outgrowth shift from the apex to subapical parts of the cell; (ii) the structural integrity of the apical MFs and the tip-to-base alignment of the CTC-calcium gradient are disturbed; and (iii) the red light induced gradient shift and the reorientation of MFs proceed in an expanded area spanning from the tip to subapical parts of the cell. Cytochalasin-D (10 g/ml) destroys the MFs. Under these conditions tip growth stops and the phototropic outgrowth is suppressed. The apical MT-structure and the CTC-calcium gradient are not influenced by the agent. Unilateral red light still induces the light-directed translocation of the gradient. Tip cells memorize a unilateral irradiation applied during growth inhibition with cytochalasin-D. After recovery in darkness the cells start to grow in the former light direction. The restoration of the MFs precedes the outgrowth. The structural alignment of the rebuilt actin bundles indicates the future growth direction. The calcium channel blocker nifedipine (10 M) also inhibits tip growth and concurrently phototropic outgrowth. Nifedipine destroys the CTC-calcium gradient and apical MFs; MTs are not influenced by the channel blocker.Abbreviations CTC chlortetracycline - DIC differential interference contrast - DMSO dimethyl sulfoxide - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N-N-tetraacetic acid - MBS 3-maleimidobenzoic acid N-hydroxysuccimide ester - MF microfilament - MT microtubule - MTSB microtubule stabilizing buffer - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - RP rhodamine labeled phalloidin     

Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function.

We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8. Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end. In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity). The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure. The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold. The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data. However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin receptors in syncytitrophoblast and fetal endothelium of human placenta. Immunohistochemical evidence for developmental changes in distribution pattern

The localisation of insulin receptors (IR) was investigated on cryosections of human non-pathologic first trimester and full term placentae by indirect immunohistochemistry with three different monoclonal antibodies (MABS). In placentae from 6 to 10 weeks post-menstruation (p-m.), only syncytiotrophoblast was stained, predominantly that of mesenchymal villi and syncytial sprouts, which are areas of high proliferative activity. In placentae from 11 to 14 weeks p-m., endothelial cells commenced to react with the IR MABS and the syncytiotrophoblast was less intensely labelled than at weeks 6 to 10 p-m. In term placentae, the microvillous membrane of the syncytiotrophoblast showed only patches of weak immunoreactivity. In contrast, the endothelial cells in the placenta but not in the umbilical cord were strongly stained. The amniotic epithelium in the chorionic plate and fibroblasts in the stroma were conspicuously labelled. The data indicate: (1) the receptor density on villous syncytiotrophoblast decreases and that of fetal endothelium increases throughout gestation; (2) syncytiotrophoblast of human term placentae expresses a low level per unit area of surface IR; and (3) the majority of IR in human term placentae is located in fetal endothelium. Apart from yet unknown functional effects of maternal and fetal insulin at the placental barrier, the results suggest a growth promoting effect on the trophoblast of maternal insulin in first trimester as well as developmental effects of fetal insulin on the feto-placental vessels at term.     

TCA-100 tumour chemosensitivity assay: Differences in sensitivity between cultured tumour cell lines and clinical studies

The BATLE LE TCA-100 tumour chemosensitivity assay has been used to evaluate chemotherapeutic drug sensitivity of cultured tumour cell lines. Studies were performed using test drug concentrations calibrated to discriminate sensitivity and resistance of clinical specimens. Strong sensitivity which appeared to be inconsistent with clinical experience was detected for some drugs and cell lines. Findings of strong sensitivity were consistent with basic differences between sensitivity testing cultured cell lines and clinical specimens. Results with cell lines frequently may not apply directly to clinical applications. Characterization of differences between cell lines and clinical specimens may assist in application of cell line findings to clinical trials.     

A Computer Simulation Study of Vntr Population Genetics: Constrained Recombination Rules Out the Infinite Alleles Model

Extensive allelic diversity in variable numbers of tandem repeats (VNTRs) has been discovered in the human genome. For population genetic studies of VNTRs, such as forensic applications, it is important to know whether a neutral mutation-drift balance of VNTR polymorphism can be represented by the infinite alleles model. The assumption of the infinite alleles model that each new mutant is unique is very likely to be violated by unequal sister chromatid exchange (USCE), the primary process believed to generate VNTR mutants. We show that increasing both mutation rates and misalignment constraint for intrachromosomal recombination in a computer simulation model reduces simulated VNTR diversity below the expectations of the infinite alleles model. Maximal constraint, represented as slippage of single repeats, reduces simulated VNTR diversity to levels expected from the stepwise mutation model. Although misalignment rule is the more important variable, mutation rate also has an effect. At moderate rates of USCE, simulated VNTR diversity fluctuates around infinite alleles expectation. However, if rates of USCE are high, as for hypervariable VNTRs, simulated VNTR diversity is consistently lower than predicted by the infinite alleles model. This has been observed for many VNTRs and accounted for by technical problems in distinguishing alleles of neighboring size classes. We use sampling theory to confirm the intrinsically poor fit to the infinite alleles model of both simulated VNTR diversity and observed VNTR polymorphisms sampled from two Papua New Guinean populations.     

Gel retardation analysis of E. coli M1 RNA-tRNA complexes.

We have analyzed complexes between tRNA and E. coli M1 RNA by electrophoresis in non-denaturing polyacrylamide gels. The RNA subunit of E. coli RNase P formed a specific complex with mature tRNA molecules. A derivative of the tRNA(Gly), endowed with the intron of yeast tRNA(ile) (60 nt), was employed to improve separation of complexed and unbound M1 RNA. Binding assays with tRNA(Gly) and intron-tRNA(Gly) as well as analysis of intron-tRNA/M1 RNA complexes on denaturing gels showed that one tRNA is bound per molecule of M1 RNA. A tRNA carrying a truncation as small as the 5'-nucleotide had a strongly reduced affinity to M1 RNA and was also a weak competitor in the cleavage reaction, suggesting that nucleotide +1 is a major determinant of tRNA recognition and that the thermodynamically stable tRNA-M1 RNA complex is relevant for enzyme function. Binding was shown to be dependent on the M1 RNA concentration in a cooperative fashion. Only a fraction of M1 RNAs (50-60%) readily formed a complex with intron-tRNA(Gly), indicating that distinct conformational subpopulations of M1 RNA may exist. Formation of the M1 RNA-tRNA(Gly), complex was very similar at 100 mM Mg++ and Ca++, corroborating earlier data that Ca++ is competent in promoting M1 RNA folding and tRNA binding. Determination of apparent equilibrium constants (app Kd) for tRNA(Gly) as a function of the Mg++ concentration supports an uptake of at least two additional Mg++ ions upon complex formation. At 20-30 mM Mg++, highest cleavage rates but strongly reduced complex formation were observed. This indicates that tight binding of the tRNA to the catalytic RNA at higher magnesium concentrations retards product release and therefore substrate turnover.