Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

Novel gene conversion between X-Y homologues located in the nonrecombining region of the Y chromosome in Felidae (Mammalia)

Genes located on the mammalian Y chromosome outside of the pseudoautosomal region do not recombine with those on the X and are predicted to either undergo selection for male function or gradually degenerate because of an accumulation of deleterious mutations. Here, phylogenetic analyses of X-Y homologues, Zfx and Zfy, among 26 felid species indicate two ancestral episodes of directed genetic exchange (ectopic gene conversion) from X to Y: once during the evolution of pallas cat and once in a common predecessor of ocelot lineage species. Replacement of the more rapidly evolving Y homologue with the evolutionarily constrained X copy may represent a mechanism for adaptive editing of functional genes on the nonrecombining region of the mammalian Y chromosome.

A synthetic cryIC gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation–PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01–0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.

Identification of genes induced by factor deprivation in hematopoietic cells undergoing apoptosis using gene-trap mutagenesis and site-specific recombination

A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/loxP) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus–thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.

Adaptive evolution of a candidate gene for aging in Drosophila

Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah (mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence at mth in population level samples of Drosophila melanogaster, D. simulans, and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.

Male fitness increases when females are eliminated from gene pool: Implications for the Y chromosome

Because the two sexes share a common gene pool while performing many different biological functions, mutations benefiting one sex may not accumulate due to counter selection in the other sex. In these experiments 99% of a haploid genome of Drosophila melanogaster was constrained to segregate like a male-limited Y chromosome for 41 generations, thereby eliminating potential counter selection in females. The synthetic Y chromosomes rapidly accumulated genetic variation that increased male fitness and decreased female fitness. The survival and fertility of females declined when they were mated to males expressing the synthetic Y chromosomes. These results suggests that opposing selection between the sexes may substantially interfere with sex-specific adaptation. They also demonstrate how intersexual evolutionary conflict can lead to perpetual degeneration of the Y via genetic hitchhiking of deleterious mutations.

Replicational and transcriptional selection on codon usage in Borrelia burgdorferi

With more than 10 fully sequenced, publicly available prokaryotic genomes, it is now becoming possible to gain useful insights into genome evolution. Before the genome era, many evolutionary processes were evaluated from limited data sets and evolutionary models were constructed on the basis of small amounts of evidence. In this paper, I show that genes on the Borrelia burgdorferi genome have two separate, distinct, and significantly different codon usages, depending on whether the gene is transcribed on the leading or lagging strand of replication. Asymmetrical replication is the major source of codon usage variation. Replicational selection is responsible for the higher number of genes on the leading strands, and transcriptional selection appears to be responsible for the enrichment of highly expressed genes on these strands. Replicational–transcriptional selection, therefore, has an influence on the codon usage of a gene. This is a new paradigm of codon selection in prokaryotes.

Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1–1, mab2–1, and mab3–1 (for maintenance of BMV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic RNA replication steps were defective. mab1–1, mab2–1, and mab3–1 slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects, mab1–1 and mab2–1 blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas mab3–1 had elevated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulation of 2a.

Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library

We have developed a strategy for the identification of peptides able to functionally replace a zinc finger domain in a transcription factor. This strategy could have important ramifications for basic research on gene regulation and for the development of therapeutic agents. In this study in yeast, we expressed chimeric proteins that included a random peptide combinatorial library in association with two zinc finger domains and a transactivating domain. The library was screened for chimeric proteins capable of activating transcription from a target sequence in the upstream regulatory regions of selectable or reporter genes. In a screen of approximately 1.5 × 107 transformants we identified 30 chimeric proteins that exhibited transcriptional activation, some of which were able to discriminate between wild-type and mutant DNA targets. Chimeric library proteins expressed as glutathione S-transferase fusions bound to double-stranded oligonucleotides containing the target sequence, suggesting that the chimeras bind directly to DNA. Surprisingly, none of the peptides identified resembled a zinc finger or other well-known transcription factor DNA binding domain.

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.

Evidence that ultraviolet markings are associated with patterns of molecular gene flow

Recent studies have shown UV vision and markings to be important in vertebrates, particularly birds, where behavioral experiments have demonstrated its potential importance in sexual selection. However, there has been no genetic evidence that UV markings determine patterns of evolution among natural populations. Here we report molecular evidence that UV markings are associated with the pattern of gene flow in the Tenerife lizard (Gallotia galloti). This species has vicariance-induced, approximate east–west lineages in Tenerife closely congruent with the primary lineages of the sympatric gecko species. Against expectations, these molecular phylogeographic lineages (representing geological history) and isolation-by-distance do not appear to influence gene flow. Sexually mature males from populations either side of a latitudinal ecotone have different UV markings and gene flow appears to be linked to this difference in UV markings. It may be that these groups with different UV sexual markings mate assortatively, restricting the gene flow between them. This has implications for debate on the relative importance of vicariance and biotopes in influencing biodiversity, with this evidence supporting the latter.

Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer

Although the systemic administration of a number of different gene products has been shown to result in the inhibition of angiogenesis and tumor growth in different animal tumor models, the relative potency of those gene products has not been studied rigorously. To address this issue, recombinant adenoviruses encoding angiostatin, endostatin, and the ligand-binding ectodomains of the vascular endothelial growth factor receptors Flk1, Flt1, and neuropilin were generated and used to systemically deliver the different gene products in several different preexisting murine tumor models. Single i.v. injections of viruses encoding soluble forms of Flk1 or Flt1 resulted in ≈80% inhibition of preexisting tumor growth in murine models involving both murine (Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic carcinoma) tumors. In contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. A strong correlation was observed between the effects of the different viruses on tumor growth and the activity of the viruses in the inhibition of corneal micropocket angiogenesis. These data underscore the need for comparative analyses of different therapeutic approaches that target tumor angiogenesis and provide a rationale for the selection of specific antiangiogenic gene products as lead candidates for use in gene therapy approaches aimed at the treatment of malignant and ocular disorders.

Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene

We have shown that the DNA demethylation complex isolated from chicken embryos has a G⋅T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a β-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor α. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

Gene genealogies and population variation in plants

Early in the development of plant evolutionary biology, genetic drift, fluctuations in population size, and isolation were identified as critical processes that affect the course of evolution in plant species. Attempts to assess these processes in natural populations became possible only with the development of neutral genetic markers in the 1960s. More recently, the application of historically ordered neutral molecular variation (within the conceptual framework of coalescent theory) has allowed a reevaluation of these microevolutionary processes. Gene genealogies trace the evolutionary relationships among haplotypes (alleles) with populations. Processes such as selection, fluctuation in population size, and population substructuring affect the geographical and genealogical relationships among these alleles. Therefore, examination of these genealogical data can provide insights into the evolutionary history of a species. For example, studies of Arabidopsis thaliana have suggested that this species underwent rapid expansion, with populations showing little genetic differentiation. The new discipline of phylogeography examines the distribution of allele genealogies in an explicit geographical context. Phylogeographic studies of plants have documented the recolonization of European tree species from refugia subsequent to Pleistocene glaciation, and such studies have been instructive in understanding the origin and domestication of the crop cassava. Currently, several technical limitations hinder the widespread application of a genealogical approach to plant evolutionary studies. However, as these technical issues are solved, a genealogical approach holds great promise for understanding these previously elusive processes in plant evolution.