De novo truncation of chromosome 16p and healing with (TTAGGG)n in the alpha-thalassemia/mental retardation syndrome (ATR-16).

We have previously described a series of patients in whom the deletion of 1-2 megabases (Mb) of DNA from the tip of the short arm of chromosome 16 (band 16p13.3) is associated with alpha-thalassemia/mental retardation syndrome (ATR-16). We now show that one of these patients has a de novo truncation of the terminal 2 Mb of chromosome 16p and that telomeric sequence (TTAGGG)n has been added at the site of breakage. This suggests that the chromosomal break, which is paternal in origin and which probably arose at meiosis, has been stabilized in vivo by the direct addition of the telomeric sequence. Sequence comparisons of this breakpoint with that of a previously described chromosomal truncation (alpha alpha)TI do not reveal extensive sequence homology. However, both breakpoints show minimal complementarity (3-4 bp) to the proposed RNA template of human telomerase at the site at which telomere repeats have been added. Unlike previously characterized individuals with ATR-16, the clinical features of this patient appear to be solely due to monosomy for the terminal portion of 16p13.3. The identification of further patients with "pure" monosomy for the tip of chromosome 16p will be important for defining the loci contributing to the phenotype of this syndrome.     

PHYLOGEOGRAPHIC PATTERNS IN MITOCHONDRIAL DNA OF THE DESERT TORTOISE (XEROBATES AGASSIZI), AND EVOLUTIONARY RELATIONSHIPS AMONG THE NORTH AMERICAN GOPHER TORTOISES

Restriction-fragment polymorphisms in mitochondrial DNA (mtDNA) were used to evaluate population-genetic structure in the desert tortoise Xerobates agassizi and to clarify evolutionary affinities among species of the gopher tortoise complex. Fourteen informative endonucleases were employed to assay mtDNAs from 56 X. agassizi representing 22 locations throughout the species' range. The mtDNA genotypes observed were readily partitioned into three major phylogenetic assemblages, each with striking geographic orientation. Overall, the X. agassizi mtDNA genotypes typify a common phylogeographic pattern, in which broad genetic uniformity of populations is interrupted by geographic features that presumably have functioned as dispersal barriers. The geologic history of the Colorado River area, which includes extensive marine incursions, may account for the marked mtDNA divergence between eastern and western X. agassizi assemblages. In mtDNA comparisons among the four species of the gopher tortoise complex, both UPGMA and Wagner parsimony analysis strongly support the recognition of two distinct species groups previously suggested by traditional systematic approaches. Furthermore, the mtDNA data identify the eastern X. agassizi assemblage as the probable inceptive lineage of X. berlandieri. Results from both intra- and interspecific comparisons illustrate how clues to historical events may be present in the geographic structure of mtDNA phylogenies.     

Solid-phase Synthesis and Cellular Localization of a C- and/or N-terminal Labelled Peptide

We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subject to further modifications including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C- and/or N-terminals. After incubation with living cultured cells these two probes were localized and it is concluded that the post-synthesis modifications can strongly modify the localization of the peptide.     

Quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus.

To compare the requirements for paramyxovirus-mediated cell fusion, the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of simian virus 5 (SV5), human parainfluenza virus 3 (HPIV-3), and Newcastle disease virus (NDV) were expressed individually or coexpressed in either homologous or heterologous combinations in CV-1 or HeLa-T4 cells, using the vaccinia virus-T7 polymerase transient expression system. The contribution of individual glycoproteins in virus-induced membrane fusion was examined by using a quantitative assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecyl rhodamine (R18) and a quantitative assay for content mixing based on the cytoplasmic activation of a reporter gene, beta-galactosidase. In these assays, expression of the individual F glycoproteins did not induce significant levels of cell fusion and no cell fusion was observed in experiments when cells individually expressing homologous F or HN proteins were mixed. However, coexpression of homologous F and HN glycoproteins resulted in extensive cell fusion. The kinetics of fusion were found to be very similar for all three paramyxoviruses studied. With NDV and HPIV-3, no cell fusion was detected when F proteins were coexpressed with heterologous HN proteins or influenza virus hemagglutinin (HA). In contrast, SV5 F protein exhibited a considerable degree of fusion activity when coexpressed with either NDV or HPIV-3 HN or with influenza virus HA, although the kinetics of fusion were two- to threefold higher when the homologous SV5 F and HN proteins were coexpressed. Thus, these data indicate that among the paramyxoviruses tested, SV5 has different requirements for cell fusion.     

Activation of ryanodine receptors by flash photolysis of caged Ca2+.

Flash photolysis of DM-nitrophen generates an extremely large [Ca2+] transient ("Ca2+ spike") at the start of each Ca2+ "step." The Ca2+ spike greatly increases the speed of activation of the ryanodine receptor channel ("supercharging") and could be responsible for apparent channel adaptation.     

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.