Selection for genes encoding secreted proteins and receptors.

Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries.

Fusion phage libraries expressing single-chain Fv antibodies were constructed from the peripheral blood lymphocytes of two melanoma patients who had been immunized with autologous melanoma cells transduced the gamma-interferon gene to enhance immunogenicity, in a trial conducted at another institution. Anti-melanoma antibodies were selected from each library by panning the phage against live cultures of the autologous tumor. After two or three rounds of panning, clones of the phage were tested by ELISA for binding to the autologous tumor cells; > 90% of the clones tested showed a strong ELISA reaction, demonstrating the effectiveness of the panning procedure for selecting antimelanoma antibodies. The panned phage population was extensively absorbed against normal melanocytes to enrich for antibodies that react with melanoma cells but not with melanocytes. The unabsorbed phage were cloned, and the specificities of the expressed antibodies were individually tested by ELISA with a panel of cultured human cells. The first tests were done with normal endothelial and fibroblast cells to identify antibodies that do not react, or react weakly, with two normal cell types, indicating some degree of specificity for melanoma cells. The proportion of phage clones expressing such antibodies was approximately 1%. Those phage were further tested by ELISA with melanocytes, several melanoma lines, and eight other tumor lines, including a glioma line derived from glial cells that share a common lineage with melanocytes. The ELISA tests identified three classes of anti-melanoma antibodies, as follows: (i) a melanoma-specific class that reacts almost exclusively with the melanoma lines; (ii) a tumor-specific class that reacts with melanoma and other tumor lines but does not react with the normal melanocyte, endothelial and fibroblast cells; and (iii) a lineage-specific class that reacts with the melanoma lines, melanocytes, and the glioma line but does not react with the other lines. These are rare classes from the immunized patients' repertoires of anti-melanoma antibodies, most of which are relatively nonspecific anti-self antibodies. The melanoma-specific class was isolated from one patient, and the lineage-specific class was isolated from the other patient, indicating that different patients can have markedly different responses to the same immunization protocol. The procedures described here can be used to screen the antibody repertoire of any person with cancer, providing access to an enormous untapped pool of human monoclonal anti-tumor antibodies with clinical and research potential.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

T cell positive selection by a high density, low affinity ligand

Interaction of the αβ T cell receptor (TCR) with major histocompatibility (MHC) molecules occupied with any of a large collection of peptides derived from self proteins is a critical step in driving T cell “positive” selection in the thymus. Interaction with this same pool of self-peptide/MHC ligands deletes T cells with potential self-reactivity. To examine how T cells survive both of these processes to form a self-tolerant mature repertoire, mice were constructed whose entire class II MHC IEk specific repertoire was positively selected on a single peptide covalently attached to the IEk molecule. In these mice T cells were identified that could respond to a variant of the positively selecting peptide bound to IEk. The affinities of the TCRs from these T cells for the positively selecting ligand were extremely low and at least 10-fold less than those for the activating ligand. These results support the theory that positive selection is driven by TCR affinities lower than those involved in T cell deletion or activation and that, if present at high concentration, even very low affinity ligands can positively select.

Evolution of foraging behavior in Drosophila by density-dependent selection

One of the rare examples of a single major gene underlying a naturally occurring behavioral polymorphism is the foraging locus of Drosophila melanogaster. Larvae with the rover allele, forR, have significantly longer foraging path lengths on a yeast paste than do those homozygous for the sitter allele, fors. These variants do not differ in general activity in the absence of food. The evolutionary significance of this polymorphism is not as yet understood. Here we examine the effect of high and low animal rearing densities on the larval foraging path-length phenotype and show that density-dependent natural selection produces changes in this trait. In three unrelated base populations the long path (rover) phenotype was selected for under high-density rearing conditions, whereas the short path (sitter) phenotype was selected for under low-density conditions. Genetic crosses suggested that these changes resulted from alterations in the frequency of the fors allele in the low-density-selected lines. Further experiments showed that density-dependent selection during the larval stage rather than the adult stage of development was sufficient to explain these results. Density-dependent mechanisms may be sufficient to maintain variation in rover and sitter behavior in laboratory populations.

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.

RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates

Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires σ factor that participates in both promoter recognition and DNA melting. RNAP lacking σ (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions. We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various σ factors. Using bubble templates containing the σD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of σ. This supports a model in which the UP element acts primarily through the α subunit of core enzyme to increase the initial association of RNAP with the promoter. Core and holoenzyme do differ substantially in the template positions chosen for initiation: σD restricts initiation to sites 8–9 nucleotides downstream of the conserved −10 element. Remarkably, σA also has a dramatic effect on start-site selection even though the σA holoenzyme is inactive on the corresponding homoduplexes. The start sites chosen by the σA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved −10 hexamer recognized by σA. Thus, σA appears to recognize the −10 region even in a single-stranded state. We propose that in addition to its described roles in promoter recognition and start-site melting, σ also localizes the transcription start site.

In vitro selection for altered divalent metal specificity in the RNase P RNA

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme’s active site.

Comparative T cell receptor repertoire selection by antigen after adoptive transfer: A glimpse at an antigen-specific preimmune repertoire

The low frequency of precursor cells specific for any particular antigen (Ag) makes it difficult to characterize preimmune T cell receptor (TCR) repertoires and to understand repertoire selection during an immune response. We have undertaken a combined adoptive transfer single-cell PCR approach to probe the Ag-specific preimmune repertoires of individual mice. Our strategy was to inject paired irradiated recipient mice with normal spleen cells prepared from individual donors and to compare the TCR repertoires subsequently selected during a CD8 response to a defined model Ag. We found that although some TCRs were shared, the TCR repertoires selected by mice receiving splenocytes from the same donor were not identical in terms of the TCRs selected and their relative frequencies. Our results together with computer simulations imply that individual mice express distinct Ag-specific preimmune TCR repertoires composed of expanded clones and that selection by Ag is a random process.

Adult fitness consequences of sexual selection in Drosophila melanogaster

Few experiments have demonstrated a genetic correlation between the process of sexual selection and fitness benefits in offspring, either through female choice or male competition. Those that have looked at the relationship between female choice and offspring fitness have focused on juvenile fitness components, rather than fitness at later stages in the life cycle. In addition, many of these studies have not controlled for possible maternal effects. To test for a relationship between sexual selection and adult fitness, we carried out an artificial selection experiment in the fruit fly, Drosophila melanogaster. We created two treatments that varied in the level of opportunity for sexual selection. Increased opportunity for female choice and male competition was genetically correlated with an increase in adult survivorship, as well as an increase in male and female body size. Contrary to previous, single-generation studies, we did not find an increase in larval competitive ability. This study demonstrates that mate choice and/or male–male competition are correlated with an increase in at least one adult fitness component of offspring.

Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein–protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.

Positive selection of an MHC class-I restricted TCR in the absence of classical MHC class I molecules

The H-2Ld alloreactive 2C T cell receptor (TCR) is commonly considered as being positively selected on the H-2Kb molecule. Surprisingly, 2C TCR+ CD8+ single-positive T cells emerge in massive numbers in fetal thymic organ culture originating from 2C transgenic, H-2KbDb−/− (2C+KbDb−/−) but not in fetal thymic organ culture from β2-microglobulin−/− 2C transgenic animals. Mature CD8+ T cells are observed in newborn but not in adult 2C+KbDb−/− mice. These CD8+ T cells express the α4β7 integrin, which allows them to populate the intestine, a pattern of migration visualized by intrathymic injection of FITC and subsequent accrual of FITC-labeled lymphocytes in the gut. We conclude that the 2C TCR is reactive not only with H-2Ld and H-2Kb, but also with nonclassical MHC class I products to enable positive selection of 2C+ T cells in the fetal and newborn thymus and to support their maintenance in the intestine.

Host-parasite dynamics and outgrowth of virus containing a single K70R amino acid change in reverse transcriptase are responsible for the loss of human immunodeficiency virus type 1 RNA load suppression by zidovudine.

The association between human immunodeficiency virus type I (HIV-1) RNA load changes and the emergence of resistant virus variants was investigated in 24 HIV-1-infected asymptomatic persons during 2 years of treatment with zidovudine by sequentially measuring serum HIV-1 RNA load and the relative amounts of HIV-1 RNA containing mutations at reverse transcriptase (RT) codons 70 (K-->R), 41 (M-->L), and 215 (T-->Y/F). A mean maximum decline in RNA load occurred during the first month, followed by a resurgence between 1 and 3 months, which appeared independent of drug-resistance. Mathematical modeling suggests that this resurgence is caused by host-parasite dynamics, and thus reflects infection of the transiently increased numbers of CD4+ lymphocytes. Between 3 and 6 months of treatment, the RNA load returned to baseline values, which was associated with the emergence of virus containing a single lysine to arginine amino acid change at RT codon 70, only conferring an 8-fold reduction in susceptibility. Despite the relative loss of RNA load suppression, selection toward mutations at RT codons 215 and 41 continued. Identical patterns were observed in the mathematical model. While host-parasite dynamics and outgrowth of low-level resistant virus thus appear responsible for the loss of HIV-1 RNA load suppression, zidovudine continues to select for alternative mutations, conferring increasing levels of resistance.

Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.

Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.

Pollen selection: a transgenic reconstruction approach.

A transgenic reconstruction experiment has been performed to determine the feasibility of male gametophytic selection to enhance transmission of genes to the next sporophytic generation. For tobacco pollen from a transgenic plant containing a single hygromycin-resistance (hygromycin phosphotransferase, hpt-) gene under control of the dc3 promoter, which is active in both sporophytic and gametophytic tissues, 3 days of in vitro maturation in hygromycin-containing medium was sufficient to result in a 50% reduction of germinating pollen, as expected for meiotic segregation of a single locus insert. Pollination of wild-type plants with the selected pollen yielded 100% transgenic offspring, as determined by the activity of the linked kanamycin-resistance gene--present within the same transferred T-DNA borders--under control of the nos promoter. This is direct proof that selection acting on male gametophytes can be a means to alter the frequency of genes in the progeny.