Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron

Recently, the 2.8 A crystal structure of one domain of the self-splicing Tetrahymena group I intron was reported. Although it revealed much about RNA tertiary interactions, it contained only half of the active site. We have now designed a series of larger molecules that contain about 70% of the intron and all of the catalytic core. These RNAs were efficient in cleavage of a substrate RNA, consisting of the approximately 100 nucleotides from the 5' end of the intron, at a site corresponding to the 5' splice site. A sparse matrix was designed specifically for large RNAs and used to screen for preliminary crystallization conditions. Of the six RNAs initially tested, five were crystallized in this initial trial. Two of these crystals were further examined. The first diffracted X-rays to only approximately 16 A resolution, even when the crystal were very large. The second diffracted as high as 3.5 A, but the crystals were twinned and therefore unusable for structural studies. Site-specific mutagenesis was performed on the latter RNA to disrupt interactions that might have been responsible for the twinning. One of these mutant RNAs produced large, single, diffraction-quality crystals. The crystals belong to the tetragonal space group P42212 and have large unit cell dimensions, a=b=178 A and c=199 A. Thus, by variation of both sequence elements and crystallization conditions, crystals of a 247 nucleotide catalytic RNA were obtained.     

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.     

Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity

The effect of genetic context on splicing of group I introns is not well understood at present. The influence of ribosomal RNA conformation on splicing of rDNA introns in vivo was investigated using a heterologous system in which the Tetrahymena group I intron is inserted into the homologous position of the Escherichia coli 23S rRNA. Mutations that block splicing in E. coli result in accumulation of unspliced 23S rRNA that is assembled into 50S complexes, but not 70S ribosomes. The data indicate that accommodation of the intron structure on the surface of the 50S subunit inhibits interactions with the small ribosomal subunit. Spliced intron RNA also remains noncovalently bound to 50S subunits on sucrose gradients. This interaction appears to be mediated by base pairing between the intron guide sequence and the 23S rRNA, because the fraction of bound intron RNA is reduced by point mutations in the IGS or deletion of the P1 helix. Association of the intron with 50S subunits correlates with slow cell growth. The results suggest that group I introns have the potential to inhibit protein synthesis in prokaryotes by direct interactions with ribosomes.     

Dynamics of time discrimination: II. The effects of multiple impulses

According to a diffusion generalization model, time discrimination is determined by the frequency and recency of preceding intervals of time. A procedure for studying rapid timing was used to investigate whether pigeons' wait-time responses were sensitive to these factors. In Experiment 1 the number (two or eight) and spacing (consecutive or far apart) of 5-s interfood intervals (called impulses) intercalated in a series of 15-s interfood intervals (nonimpulses) were studied. Experiment 2 was identical to the first but the interfood intervals were increased by a factor of three. Overall, impulses shortened wait times in the next interfood interval. However, several impulses occurring in succession extended the localized effect of an impulse: Wait times following a set of eight-close impulses were slow to recover to preimpulse levels. The results show that linear waiting is only an approximation to the dynamic process, and a process that is sensitive to events in an animal's remote past, such as the diffusion generalization model, provides a better account of rapid timing effects.     

Sequence variation of the tRNA(Leu) intron as a marker for genetic diversity and specificity of symbiotic cyanobacteria in some lichens

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNA(Leu) (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNA(Leu) (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNA(Leu) (UAA) intron can be of great value when examining cyanobacterial diversity.     

Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis

Group II introns are self-splicing RNA molecules that are of considerable interest as ribozymes, mobile genetic elements and examples of folded RNA. Although these introns are among the most common ribozymes, little is known about the chemical and structural determinants for their reactivity. By using nucleotide analog interference mapping (NAIM), it has been possible to identify the nucleotide functional groups (Rp phosphoryls, 2'-hydroxyls, guanosine exocyclic amines, adenosine N7 and N6) that are most important for composing the catalytic core of the intron. The majority of interference effects occur in clusters located within the two catalytically essential Domains 1 and 5 (D1 and D5). Collectively, the NAIM results indicate that key tetraloop-receptor interactions display a specific chemical signature, that the epsilon-epsilon' interaction includes an elaborate array of additional features and that one of the most important core structures is an uncharacterized three-way junction in D1. By combining NAIM with site-directed mutagenesis, a new tertiary interaction, kappa-kappa', was identified between this region and the most catalytically important section of D5, adjacent to the AGC triad in stem 1. Together with the known zeta-zeta' interaction, kappa-kappa' anchors D5 firmly into the D1 scaffold, thereby presenting chemically essential D5 functionalities for participation in catalysis.     

Preventing discrimination against volunteers in prophylactic HIV vaccine trials: lessons from a phase II trial

OBJECTIVE: This was a randomized, double-blind, crossover study of 30 children with attention-deficit/hyperactivity disorder (ADHD) that evaluated the time course effects of four doses of Adderall (5, 10, 15, and 20 mg), an inactive control (placebo), and a positive control (clinical dose of methylphenidate). METHOD: For each treatment condition, a capsule was administered in the morning and assessments were performed in an analog classroom setting every 1.5 hours across the day. Subjective (teacher ratings of deportment and attention) and objective (scores on math tests) measures were obtained for each classroom session, and these measures were used to evaluate time-response and dose response effects of Adderall. RESULTS: For doses of Adderall greater than 5 mg, significant time course effects were observed. Rapid improvements on teacher ratings and math performance were observed by 1.5 hours after administration, and these effects dissipated by the end of the day. The specific pattern of time course effects depended on dose: the time of peak effects and the duration of action increased with dose of Adderall. CONCLUSIONS: This documentation of efficacy in a controlled study supports the addition of Adderall to the armamentarium of psychotropic medications for the treatment of ADHD. The differences in time-response patterns of Adderall and methylphenidate may help tailor treatment to meet specific clinical needs of different children with ADHD.     

Reverse discrimination: The relationship of amount of perceived discrimination toward a minority group on the behaviour of majority group members.

Male and female members of 4 racial groups solicited charity donations from 7,120 middle-class Canadian whites in both public and private conditions. Black and Indian solicitors received significantly greater donations than white solicitors, who in turn received significantly greater donations than oriental solicitors. 1 mo. later, white interviewers asked a randomly-selected subsample (n = 500) to estimate the degree of discrimination to which various ethnic and racial groups are generally subjected. Results provide supportive evidence for 1 part of a "theory of reverse discrimination," i.e., that when middle-class whites are involved in "trivial" interactions with minority group members whom they perceive as belonging to groups that have been targets of discrimination, they will treat those minority group members better than they treat another white in identical circumstances. (French summary) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Novel therapeutic strategy for atherosclerosis: ribozyme oligonucleotides against apolipoprotein(a) selectively inhibit apolipoprotein(a) but not plasminogen gene expression

BACKGROUND: Because mechanisms of atherosclerosis by lipoprotein(a) [Lp(a)] have been postulated in the decrease in active transforming growth factor-beta conversion by decreased plasmin, selective decrease in apolipoprotein(a) [apo(a)] independent of plasminogen may have therapeutic values. Although antisense can decrease apo(a), its application may be difficult because of very high homology of apo(a) gene to plasminogen. Thus we used ribozyme strategy that actively cleaves targeted genes to selectively inhibit apo(a) expression. METHODS AND RESULTS: We constructed ribozyme oligonucleotides containing phosphorothioate DNA- and RNA-targeted kringle 4 of the apo(a) gene that showed 80% homology to plasminogen. Transfection of human apo(a) gene produced Lp(a) in medium of HepG2 cells, whereas Lp(a) could not be detected in control cells. Cotransfection of ribozyme and apo(a) gene resulted in the decrease in mRNA of apo(a) but not plasminogen. Moreover, marked decrease in Lp(a) was also observed in the medium transfected with ribozyme and apo(a) gene compared with apo(a) gene alone (P<0.01), whereas there was no significant change in plasminogen level between ribozyme-transfected and control cells. Incubation of human vascular smooth muscle cells (VSMC) with conditioned medium from apo(a)-transfected HepG2 cells resulted in a significant increase in VSMC number, whereas addition of conditioned medium from cells cotransfected with ribozyme oligonucleotides and apo(a) gene resulted in no VSMC growth (P<0.01). DNA-based control oligonucleotides and mismatched ribozyme oligonucleotides did not have an inhibitory effect on Lp(a) production. CONCLUSIONS: Overall, our data revealed that transfection of ribozyme against the apo(a) gene resulted in the selective inhibition of the apo(a) but not the plasminogen gene, providing novel therapeutic strategy for treatment of high Lp(a), a risk factor for atherosclerosis.     

The cost of search for multiple targets: Effects of practice and target similarity.

With the use of X-ray images, performance in the simultaneous search for two target categories was compared with performance in two independent searches, one for each category. In all cases, displays contained one target at most. Dual-target search, for both categories simultaneously, produced a cost in accuracy, although the magnitude of this dual-target cost was affected by the nature of the targets. When target feature sets shared values, accuracy in dual-target search was equivalent to that in the less accurate of the two single-target searches. However, when targets comprised different feature sets, accuracy in dual-target search was lower than in either single-target search. These results held after practice. In conclusion, dual-target search performance depends on the target representations required for search. When combined representations contain conflicting values within the most informative feature dimensions, then there is a cost in performance. When target representations share features, the search can be guided by the common values so that resources are not wasted on irrelevant distractors. The implication is that security screener performance might be improved by specializing in searching for threat categories that share features. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Search and retrieval patterns for performance information: Effects on evaluations of multiple targets.

Examined search, organization, and use of performance information about multiple targets. In Exp I, 41 undergraduates searched among 64 performance vignettes, stored in a 4?×?4?×?4 (Target?×?Task?×?Occasion) computer array. The predominant search pattern involved examining a set of performances by one target before proceeding to another. Exp II tested whether various acquisition patterns affect organization and use of performance information. 125 Ss were shown 16 vignettes from Exp I, organized in target-by-target (target blocked), task-by-task (task blocked), or mixed sequences. Results indicate greater recall under blocked than under mixed conditions. Blocking also influenced organization in recall. Target blocking produced clustering by target; task blocking produced clustering by task; mixed presentation produced no discernible clustering. Accuracy of ratings for overall target performance was unaffected by blocking, but task blocking produced better differentiation among instances of good and poor performance exhibited by specific targets. (22 ref) (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Trial of trazodone for posttraumatic stress disorder using a multiple baseline group design

Six patients with combat-related posttraumatic stress disorder (PTSD) entered a multiple-baseline trial of trazodone, beginning with 50 mg/day and increasing to 400 mg/day until response was maximal. Total Clinician-Administered PTSD Scale scores decreased from a mean of 92 at baseline to 79 at end point, and self-reported PTSD symptoms as measured by the Davidson Trauma Scale paralleled these results (mean of 102 at baseline to 88 at end point). Based on clinician global improvement scores, four patients were rated as much improved and two were rated to be minimally improved. Improvement in social and occupational functioning, and depression was minimal. Available follow-up scores for PTSD symptoms indicated that gains were maintained. Sleep was the first symptom to improve at 2 to 3 months. No dropouts during the treatment period occurred, and reported side effects were quite low. These preliminary data suggest that trazodone may be effective in reducing the three primary clusters of symptoms of PTSD. These findings should be confirmed by using a larger sample in a double-blind, placebo-controlled study.     

Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron PI.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.     

Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recently we developed a positive selection vector for analysis of plasmid recombination events in B. subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Several lines of evidence suggest that single-strand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resemble potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.     

Molecular basis for species and ligand specificity of a monoclonal antibody raised against human IGF-I

The anti-hIGF-I monoclonal antibody, alpha-sm1.2, was found to have substantial crossreactivity with human and rat IGF-II, but recognized rat IGF-I only when this ligand was present at very high concentration. (E50 for hIGF-I approximately 3.5 ng/tube vs. approximately 12,000 ng/tube for rat IGF-I). In the context of previous studies to define the epitope(s) of alpha-sm1.2, these findings point to the critical importance of aspartic acid at residue 20 in the B domain in determining the species and ligand specificity of this antibody. Previous studies using this antibody in rodent tissues may require reinterpretation in the light of these findings.     

Sporadic distribution of tRNA(Arg)CCU introns among alpha-purple bacteria: evidence for horizontal transmission and transposition of a group I intron

A group I intron interrupts the tRNA(Arg)CCU gene of the alpha-purple bacterium Agrobacterium tumefaciens (B. Reinhold-Hurek and D. A. Shub, Nature [London] 357:173-176, 1992). In this study, we assess the distribution of the corresponding intron among 12 additional species of alpha-purple bacteria. Of 10 newly identified tRNA(Arg)CCU genes, we found only two that contained an intron homologous to that of the Agrobacterium intron. This restricted and scattered distribution of the tRNA(Arg)CCUg intron among alpha-purple bacteria is consistent with a recent origin and horizontal transmission. Primary and secondary structural similarities between tRNA(Leu)UAA introns found in strains of the cyanobacterium Microcystis aeruginosa (K. Rudi and K. S. Jacobsen, FEMS Microbiol. Lett. 156:293-298, 1997) and alpha-purple tRNA(Arg)CCU introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA(Leu)UAA introns.     

Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer

BACKGROUND: The bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers. Towards this end, we have synthesized an anti-bcl-2 gene therapeutic reagent based on ribozyme technology and have tested its effectiveness against bcl-2 mRNA in vitro and in vivo. METHODS: A divalent hammerhead ribozyme was constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA. A disabled ribozyme lacking catalytic activity was also constructed as a control reagent for our experiments. The ribozymes were tested for endonucleolytic activity against synthetic and natural bcl-2 mRNAs. Simple transfection procedures were then utilized to introduce the ribozymes into cultured prostate cancer cells (LNCaP derivatives). We measured the effects of the ribozymes on endogenous expression of bcl-2 mRNA and protein in these cells as well as their ability to induce apoptosis. RESULTS: The functional but not the disabled ribozyme was able to rapidly degrade bcl-2 mRNA in vitro, without the requirement for any other cellular protein or factor. When directly transfected into LNCaP cell variants, it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment. This activity was sufficient to induce apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line. For the high-bcl-2-expressing variant, however, it did restore the ability to genetically respond to a secondary apoptotic agent, phorbol ester, as evidenced by the renewed ability of phorbol ester to induce NGF1A mRNA in these cells. CONCLUSIONS: This study supports the potential utility of an anti-bcl-2 ribozyme reagent for reducing or eliminating bcl-2 expression from hormone-refractory prostate cancer cells and for killing prostate cancer cells. As such, it is the first step toward an effective gene therapy against hormone-refractory human prostate cancers.