Contrast-FEL—A Test for Differences in Selective Pressures at Individual Sites among Clades and Sets of Branches

A number of evolutionary hypotheses can be tested by comparing selective pressures among sets of branches in a phylogenetic tree. When the question of interest is to identify specific sites within genes that may be evolving differently, a common approach is to perform separate analyses on subsets of sequences and compare parameter estimates in a post hoc fashion. This approach is statistically suboptimal and not always applicable. Here, we develop a simple extension of a popular fixed effects likelihood method in the context of codon-based evolutionary phylogenetic maximum likelihood testing, Contrast-FEL. It is suitable for identifying individual alignment sites where any among the K≥2 sets of branches in a phylogenetic tree have detectably different ω ratios, indicative of different selective regimes. Using extensive simulations, we show that Contrast-FEL delivers good power, exceeding 90% for sufficiently large differences, while maintaining tight control over false positive rates, when the model is correctly specified. We conclude by applying Contrast-FEL to data from five previously published studies spanning a diverse range of organisms and focusing on different evolutionary questions.     

Presence of single-stranded DNA in PCR products of slow electrophoretic mobility.

PCR products were characterized by electrophoresis, blotting and hybridization. In addition to the bands of expected size, bands of slower electrophoretic mobility were often detected. The slower bands completely disappeared when the PCR products were subjected to slow cooling, treated with S1 nuclease or run on an alkaline gel, whereas the bands of expected size were unaffected. The slower bands are therefore likely to contain single-stranded DNA.     

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase,UCH-L1

We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.     

The gene coding for the human T-lymphocyte CD2 antigen is located on chromosome 1p

Summary A cDNA clone encoding the human T lymphocyte sheep erythrocyte receptor [the CD2 (T11) antigen] was used as a probe to define the chromosomal location of the gene. The signal, revealed by hybridisation to Southern blots of genomic DNA from somatic cell hybrids, showed a high degree of concordance for human chromosome 1. In particular, the hybrid F4Sc13C19 which contained the short arm only of human chromosome 1 was positive. The location of the CD2 gene to 1p13 was confirmed by in situ hybridisation.     

The reconstitution of a hybrid histone octamer containing avian 110Cys-des-thio-histone H3 and sea-urchin 73Cys-histone H4

A hybrid histone octamer was reconstituted from erythrocyte H2A and H2B, avian [110 Cys-des-thio]histone H3 and the sea-urchin sperm [73Cys]H4 variant. [110Cys-Des-thio]histone H3 was prepared by reaction of natural H3 with Raney nickel. The ability of the hybrid octamer to crystallize to the same form as the natural octamer demonstrated that the chemical modification of cysteine to alanine in H3 and the mutation from threonine to cysteine in sperm H4 do not alter histone-histone interactions in the octamer. Since the sulfhydryl groups of both H4 molecules are fully accessible to 5,5'-dithiobis(2-nitrobenzoate) these residues provide suitable sites for the introduction of a single cysteine-specific label per H4 molecule in the octamer.     

Aberrant DNase I digestion kinetics of nucleosomal core particles from sea urchin sperm

The pancreatic deoxyribonuclease (DNase I) digestion rates at the susceptible sites on nucleosomal core particles from blastula, gastrula and sperm cells of the sea urchin, Parechinus angulosus, have been determined. Although there are differences in their isohistone composition, the rates of digestion are similar for both embryonic stages. The rates of digestion for sperm core particles are 3-5 times lower than for embryo core particles at the more, and up to 2.5 times lower at the less susceptible sites. An explanation for these differences could be sought in the sperm isohistones H2B which are characterized by N-terminal extensions of 20-25 amino acid residues.     

Pertussigen enhances antigen-driven interferon-gamma production by sensitized lymphoid cells

We have studied the effects on interferon-gamma (IFN-gamma) production of pertussigen, a protein toxin from Bordetella pertussis that augments and prolongs delayed-type hypersensitivity (DTH) reactions. Lymphoid cell suspensions from immunized mice were incubated with antigen or mitogen, and the culture supernatants were assayed for IFN-gamma. The production of IFN-gamma on exposure to specific antigen or concanavalin A was greatly enhanced if mice were given pertussigen at the time of immunization. There was no detectable IFN-gamma production when cells were exposed to saline or to an irrelevant antigen. The effect of pertussigen on antigen-driven IFN-gamma production correlated with its effect on the capacity of the same cell populations to transfer DTH. The enhanced IFN-gamma production by cells from mice given pertussigen could not be attributed to an increased antigen-presenting capacity of this cell population. Production of IFN-gamma was abolished if the cells were pretreated with emetine, but not with mitomycin C, and the release of IFN-gamma was not detected in the first 8 hr of culture. After immunization with pertussigen, IFN-gamma was produced by lymph node and spleen cells from 7 days onward and both cell types produced IFN-gamma until at least 30 days after immunization. It is suggested that the augmentation of antigen-specific IFN-gamma production may contribute to the prolonged DTH reactions induced by pertussigen in vivo.     

Covalent labelling of histones with aurothiomalate. Gold-labelled H2A-H2B dimers assemble to octamers, which form crystalline helical tubes

Histone octamers were covalently labelled with aurothiomalate at amino groups by the method of carbodiimide activation. The labelling procedure was demonstrated to result in the specific covalent coupling through a single bond of the heavy metal atom label to protein amino groups. Such octamers were dissociated to yield soluble H2A-H2B dimers containing three gold atoms per dimer. The dimers were reconstituted with native H3-H4 tetramers to form labelled octamers, which were crystallized to form helical tubes. This strongly suggests that this procedure resulted in minimal changes of protein conformation.