Selection and nuclear immobilization of exportable RNAs

The intracellular distribution of RNAs depends on interactions of cis-acting nuclear export elements or nuclear retention elements with trans-acting nuclear transport or retention factors. To learn about the relationship between export and retention, we isolated RNAs that are exported from nuclei of Xenopus laevis oocytes even when most RNA export is blocked by an inhibitor of Ran-dependent nucleocytoplasmic transport, the Matrix protein of vesicular stomatitis virus. Export of the selected RNAs is saturable and specific. When present in chimeric RNAs, the selected sequences acted like nuclear export elements in promoting efficient export of RNAs that otherwise are not exported; the pathway used for export of these chimeric RNAs is that used for the selected RNAs alone. However, these chimeric RNAs, unlike the selected RNAs, were not exported in the presence of Matrix protein; thus, the nonselected sequences can cause retention of the selected RNA sequences under conditions of impaired nucleocytoplasmic transport. We propose that most RNAs are transiently immobilized in the nucleus and that release of these RNAs is an essential and early step in export. Release correlates with functional Ran-dependent transport, and the lack of export of chimeric RNAs may result from interference with the Ran system.

Possible ecological risks of transgenic organism release when transgenes affect mating success: Sexual selection and the Trojan gene hypothesis

Widespread interest in producing transgenic organisms is balanced by concern over ecological hazards, such as species extinction if such organisms were to be released into nature. An ecological risk associated with the introduction of a transgenic organism is that the transgene, though rare, can spread in a natural population. An increase in transgene frequency is often assumed to be unlikely because transgenic organisms typically have some viability disadvantage. Reduced viability is assumed to be common because transgenic individuals are best viewed as macromutants that lack any history of selection that could reduce negative fitness effects. However, these arguments ignore the potential advantageous effects of transgenes on some aspect of fitness such as mating success. Here, we examine the risk to a natural population after release of a few transgenic individuals when the transgene trait simultaneously increases transgenic male mating success and lowers the viability of transgenic offspring. We obtained relevant life history data by using the small cyprinodont fish, Japanese medaka (Oryzias latipes) as a model. Our deterministic equations predict that a transgene introduced into a natural population by a small number of transgenic fish will spread as a result of enhanced mating advantage, but the reduced viability of offspring will cause eventual local extinction of both populations. Such risks should be evaluated with each new transgenic animal before release.

In vitro selection and evolution of functional proteins by using ribosome display

We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 108-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.

Is sexual selection and species recognition a continuum? Mating behavior of the stalk-eyed fly Drosophila heteroneura

If behavioral isolation between species can evolve as a consequence of sexual selection within a species, then traits that are both sexually selected and used as a criterion of species recognition by females should be identifiable. The broad male head of the Hawaiian picture-winged fly Drosophila heteroneura is a novel sexual dimorphism that may be sexually selected and involved in behavioral isolation from D. silvestris. We found that males with broad heads are more successful in sexual selection, both through female mate choice and through aggressive interactions. However, female D. heteroneura do not discriminate against hybrids on the basis of their head width. Thus, this novel trait is sexually selected but is not a major contributor to species recognition. Our methods should be applicable to other species in which behavioral isolation is a factor.

Tuning of activation thresholds explains flexibility in the selection and development of T cells in the thymus

Immature CD4+CD8+ thymocytes expressing T-cell antigen receptors (TCR) are selected by TCR-mediated recognition of peptides associated with major histocompatibility complex molecules on thymic stromal cells. Selection ensures reactivity of the mature cells to foreign antigens and tolerance to self. Although much has been learned about the factors that determine whether a thymocyte with a given specificity will be positively or negatively selected, selection as an aspect of the developmental process as a whole is less well-understood. Here we invoke a model in which thymocytes tune their response characteristics individually and dynamically in the course of development. Cellular development and selection are driven by receptor-mediated metabolic perturbations. Perturbation is a measure of the net intracellular change induced by external stimulation. It results from the integration of several signals and countersignals over time and therefore depends on the environment and the maturation stage of the cell. Individual cell adaptation limits the range of perturbations. Such adaptation renders thymocytes less sensitive to the level of stimulation per se, but responsive to environmental changes in that level. This formulation begins to explain the mechanisms that link developmental and selection events to each other.

Dissociation of response conflict, attentional selection, and expectancy with functional magnetic resonance imaging

Two different attentional networks have been associated with visuospatial attention and conflict resolution. In most situations either one of the two networks is active or both are increased in activity together. By using functional magnetic resonance imaging and a flanker task, we show conditions in which one network (anterior attention system) is increased in activity whereas the other (visuospatial attention system) is reduced, showing that attentional conflict and selection are separate aspects of attention. Further, we distinguish between neural systems involved in different forms of conflict. Specifically, we dissociate patterns of activity in the basal ganglia and insula cortex during simple violations in expectancies (i.e., sudden changes in the frequency of an event) from patterns of activity in the anterior attention system specifically correlated with response conflict as evidenced by longer response latencies and more errors. These data provide a systems-level approach in understanding integrated attentional networks.

Purifying selection and birth-and-death evolution in the ubiquitin gene family

Ubiquitin is a highly conserved protein that is encoded by a multigene family. It is generally believed that this gene family is subject to concerted evolution, which homogenizes the member genes of the family. However, protein homogeneity can be attained also by strong purifying selection. We therefore studied the proportion (pS) of synonymous nucleotide differences between members of the ubiquitin gene family from 28 species of fungi, plants, and animals. The results have shown that pS is generally very high and is often close to the saturation level, although the protein sequence is virtually identical for all ubiquitins from fungi, plants, and animals. A small proportion of species showed a low level of pS values, but these values appeared to be caused by recent gene duplication. It was also found that the number of repeat copies of the gene family varies considerably with species, and some species harbor pseudogenes. These observations suggest that the members of this gene family evolve almost independently by silent nucleotide substitution and are subjected to birth-and-death evolution at the DNA level.

Altered thymic positive selection and intracellular signals in Cbl-deficient mice

Cbl is the product of the protooncogene c-cbl and is involved in T cell antigen receptor (TCR)-mediated signaling. To understand the role of Cbl for immune system development and function, we generated a Cbl-deficient mouse strain. In Cbl-deficient mice, positive selection of the thymocytes expressing major histocompatibility complex class II-restricted transgenic TCR was significantly enhanced. Two factors may have contributed to the altered thymic selection. First, Cbl deficiency markedly up-regulated the activity of ZAP-70 and mitogen-activated protein kinases. The mitogen-activated protein kinase pathway was shown previously to be involved in thymic positive selection. Second, Cbl-deficient thymocytes expressed CD3 and CD4 molecules at higher levels, which consequently may increase the avidity of TCR/major histocompatibility complex/coreceptor interaction. Thus, Cbl plays a novel role in modulating TCR-mediated multiple signaling pathways and fine-tunes the signaling threshold for thymic selection.

Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration

We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.

Positive selection and rates of evolution in immunodeficiency viruses from humans and chimpanzees.

Evolutionary theory predicts the recent spread of primate immunodeficiency viruses (PIVs) to new human populations to be accompanied by positive selection in response to new host environments and/or by random genetic drift. I assess evidence for positive selection in human and chimpanzee PIVs type I (PIV1s), using ratios of synonymous to nonsynonymous nucleotide change based on branch lengths and outgroup rooting. Ratios are smaller for PIV1s from humans than for PIV1 from a chimpanzee for the pol, gag, and env glycoprotein 120 (gp120) regions, indicating greater effects of positive selection in PIV1s from humans. Parsimony-based relative rate tests for amino acid changes showed significant differences between PIV1s from humans and chimpanzees in 18 of 48 pairwise comparisons, with all 18 showing faster rates of change in PIV1s from humans. This study indicates that in some instances, the recent evolution of human PIV1s follows a speciational pattern, in which increased diversification of taxa is correlated with greater amounts of character change appearing and being maintained through time. This extends the generality of the speciational pattern to a group of organisms (viruses) having the fastest known rates of anagenetic change for nucleotide characters and indicates that comprehensive understanding of PIV1 evolution requires consideration of both anagenetic change within viral lineages and the relative historical success of different viral clades. Phylogenetic analyses show that neither PIV1s infecting humans nor those infecting chimpanzees represent monophyletic groups and suggest multiple host-species shifts for PIV1s.

Sequence specificity of triplex DNA formation: Analysis by a combinatorial approach, restriction endonuclease protection selection and amplification.

We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.

A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3′ splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3′ splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5′ region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54–55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3′ end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.

RNA-peptide fusions for the in vitro selection of peptides and proteins

Covalent fusions between an mRNA and the peptide or protein that it encodes can be generated by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. The stable linkage between the informational (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. Fusions between a synthetic mRNA and its encoded myc epitope peptide have been enriched from a pool of random sequence mRNA-peptide fusions by immunoprecipitation. Covalent RNA-peptide fusions should provide an additional route to the in vitro selection and directed evolution of proteins.