Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci

DsrA is an 87-nt untranslated RNA that regulates both the global transcriptional silencer and nucleoid protein H-NS and the stationary phase and stress response sigma factor RpoS (σs). We demonstrate that DsrA acts via specific RNA:RNA base pairing interactions at the hns locus to antagonize H-NS translation. We also give evidence that supports a role for RNA:RNA interactions at the rpoS locus to enhance RpoS translation. Negative regulation of hns by DsrA is achieved by the RNA:RNA interaction blocking translation of hns RNA. In contrast, results suggest that positive regulation of rpoS by DsrA occurs by formation of an RNA structure that activates a cis-acting translational operator. Sequences within DsrA complementary to three additional genes, argR, ilvIH, and rbsD, suggest that DsrA is a riboregulator of gene expression that acts coordinately via RNA:RNA interactions at multiple loci.

Identification of genes required for Drosophila eye development using a phenotypic enhancer-trap

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (i) the isolation of enhancer-traps with a specific expression pattern, and (ii) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.

Screening for imprinted genes by allelic message display: Identification of a paternally expressed gene Impact on mouse chromosome 18

A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes. In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F1 hybrids and pooled backcross progenies. From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified. The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function. In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages. Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.

Three novel families of miniature inverted-repeat transposable elements are associated with genes of the yellow fever mosquito, Aedes aegypti

Three novel families of transposable elements, Wukong, Wujin, and Wuneng, are described in the yellow fever mosquito, Aedes aegypti. Their copy numbers range from 2,100 to 3,000 per haploid genome. There are high degrees of sequence similarity within each family, and many structural but not sequence similarities between families. The common structural characteristics include small size, no coding potential, terminal inverted repeats, potential to form a stable secondary structure, A+T richness, and putative 2- to 4-bp A+T-biased specific target sites. Evidence of previous mobility is presented for the Wukong elements. Elements of these three families are associated with 7 of 16 fully or partially sequenced Ae. aegypti genes. Characteristics of these mosquito elements indicate strong similarities to the miniature inverted-repeat transposable elements (MITEs) recently found to be associated with plant genes. MITE-like elements have also been reported in two species of Xenopus and in Homo sapiens. This characterization of multiple families of highly repetitive MITE-like elements in an invertebrate extends the range of these elements in eukaryotic genomes. A hypothesis is presented relating genome size and organization to the presence of highly reiterated MITE families. The association of MITE-like elements with Ae. aegypti genes shows the same bias toward noncoding regions as in plants. This association has potentially important implications for the evolution of gene regulation.

Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive “glycomodules” extensively O-glycosylated through the Hyp residues. The patterns of Hyp-O-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1–5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test, we designed two simple HRGPs as fusion proteins with green fluorescent protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.

Protective anti-tumor immunity induced by vaccination with recombinant adenoviruses encoding multiple tumor-associated cytotoxic T lymphocyte epitopes in a string-of-beads fashion

Vaccines harboring genes that encode functional oncoproteins are intrinsically hazardous, as their application may lead to introduction of these genes into normal cells and thereby to tumorigenesis. On the other hand, oncoproteins are especially attractive targets for immunotherapy of cancer, as their expression is generally required for tumor growth, making the arisal of tumor variants lacking these antigens unlikely. Using murine tumor models, we investigated the efficacy of polyepitope recombinant adenovirus (rAd) vaccines, which encode only the immunogenic T cell epitopes derived from several oncogenes, for the induction of protective anti-tumor immunity. We chose to employ rAd, as these are safe vectors that do not induce the side effects associated with, for example, vaccinia virus vaccines. A single polyepitope rAd was shown to give rise to presentation of both H-2 and human leukocyte antigen-restricted cytotoxic T lymphocyte (CTL) epitopes. Moreover, vaccination with a rAd encoding H-2-restricted CTL epitopes, derived from human adenovirus type 5 early region 1 and human papilloma virus type 16-induced tumors, elicited strong tumor-reactive CTL and protected the vaccinated animals against an otherwise lethal challenge with either of these tumors. The protection induced was superior compared with that obtained by vaccination with irradiated tumor cells. Thus, vaccination with polyepitope rAd is a powerful approach for the induction of protective anti-tumor immunity that allows simultaneous immunization against multiple tumor-associated T cell epitopes, restricted by various major histocompatibility complex haplotypes.

Xenopus Kielin: A dorsalizing factor containing multiple chordin-type repeats secreted from the embryonic midline

The midline tissues are important inductive centers of early vertebrate embryos. By signal peptide selection screening, we isolated a secreted factor, Kielin, which contains multiple cys-rich repeats similar to those in chordin (Chd). Expression of Kielin starts at midgastrula stages in the notochord and is detected in the floor plate of neurula embryos. Kielin is induced in mesoderm and in ectoderm by nodal-related genes. Chd is sufficient to activate Kielin expression in mesoderm whereas Shh or HNF-3β in addition to Chd is required for induction in ectoderm. Kielin has a distinct biological activity from that of Chd. Injection of Kielin mRNA causes dorsalization of ventral marginal zone explants and expansion of MyoD expression in neurula embryos. Unlike Chd, Kielin does not efficiently induce neural differentiation of animal cap ectoderm, suggesting that the activity of Kielin is not simply caused by BMP4 blockade. Kielin is a signaling molecule that mediates inductive activities of the embryonic midline.

Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products

The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds—“unnatural” natural products—by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.

Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling

Neurotrophic factors such as nerve growth factor (NGF) promote a wide variety of responses in neurons, including differentiation, survival, plasticity, and repair. Such actions often require changes in gene expression. To identify the regulated genes and thereby to more fully understand the NGF mechanism, we carried out serial analysis of gene expression (SAGE) profiling of transcripts derived from rat PC12 cells before and after NGF-promoted neuronal differentiation. Multiple criteria supported the reliability of the profile. Approximately 157,000 SAGE tags were analyzed, representing at least 21,000 unique transcripts. Of these, nearly 800 were regulated by 6-fold or more in response to NGF. Approximately 150 of the regulated transcripts have been matched to named genes, the majority of which were not previously known to be NGF-responsive. Functional categorization of the regulated genes provides insight into the complex, integrated mechanism by which NGF promotes its multiple actions. It is anticipated that as genomic sequence information accrues the data derived here will continue to provide information about neurotrophic factor mechanisms.

Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics

The evolutionary relationships of 46 Shigella strains representing each of the serotypes belonging to the four traditional Shigella species (subgroups), Dysenteriae, Flexneri, Boydii, and Sonnei, were determined by sequencing of eight housekeeping genes in four regions of the chromosome. Analysis revealed a very similar evolutionary pattern for each region. Three clusters of strains were identified, each including strains from different subgroups. Cluster 1 contains the majority of Boydii and Dysenteriae strains (B1–4, B6, B8, B10, B14, and B18; and D3–7, D9, and D11–13) plus Flexneri 6 and 6A. Cluster 2 contains seven Boydii strains (B5, B7, B9, B11, B15, B16, and B17) and Dysenteriae 2. Cluster 3 contains one Boydii strain (B12) and the Flexneri serotypes 1–5 strains. Sonnei and three Dysenteriae strains (D1, D8, and D10) are outside of the three main clusters but, nonetheless, are clearly within Escherichia coli. Boydii 13 was found to be distantly related to E. coli. Shigella strains, like the other pathogenic forms of E. coli, do not have a single evolutionary origin, indicating convergent evolution of Shigella phenotypic properties. We estimate the three main Shigella clusters to have evolved within the last 35,000 to 270,000 years, suggesting that shigellosis was one of the early infectious diseases of humans.

Normal cardiovascular development in mice deficient for 16 genes in 550 kb of the velocardiofacial/ DiGeorge syndrome region

Hemizygous interstitial deletions in human chromosome 22q11 are associated with velocardiofacial syndrome and DiGeorge syndrome and lead to multiple congenital abnormalities, including cardiovascular defects. The gene(s) responsible for these disorders is thought to reside in a 1.5-Mb region of 22q11 in which 27 genes have been identified. We have used Cre-mediated recombination of LoxP sites in embryonic stem cells and mice to generate a 550-kb deletion encompassing 16 of these genes in the corresponding region on mouse chromosome 16. Mice heterozygous for this deletion are normal and do not exhibit cardiovascular abnormalities. Because mice with a larger deletion on mouse chromosome 16 do have heart defects, the results allow us to exclude these 16 genes as being solely, or in combination among themselves, responsible for the cardiovascular abnormalities in velocardiofacial/DiGeorge syndrome. We also generated mice with a duplication of the 16 genes that may help dissect the genetic basis of “cat eye” and derivative 22 syndromes that are characterized by extra copies of portions of 22q11, including these 16 genes. We also describe a strategy for selecting cell lines with defined chromosomal rearrangements. The method is based on reconstitution of a dominant selection marker after Cre-mediated recombination of LoxP sites. Therefore it should be widely applicable to many cell lines.

Nonsynonymous substitution in abalone sperm fertilization genes exceeds substitution in introns and mitochondrial DNA

Strong positive Darwinian selection acts on two sperm fertilization proteins, lysin and 18-kDa protein, from abalone (Haliotis). To understand the phylogenetic context for this dramatic molecular evolution, we obtained sequences of mitochondrial cytochrome c oxidase subunit I (mtCOI), and genomic sequences of lysin, 18-kDa, and a G protein subunit. Based on mtDNA differentiation, four north Pacific abalone species diverged within the past 2 million years (Myr), and remaining north Pacific species diverged over a period of 4–20 Myr. Between-species nonsynonymous differences in lysin and 18-kDa exons exceed nucleotide differences in introns by 3.5- to 24-fold. Remarkably, in some comparisons nonsynonymous substitutions in lysin and 18-kDa genes exceed synonymous substitutions in mtCOI. Lysin and 18-kDa intron/exon segments were sequenced from multiple red abalone individuals collected over a 1,200-km range. Only two nucleotide changes and two sites of slippage variation were detected in a total of >29,000 nucleotides surveyed. However, polymorphism in mtCOI and a G protein intron was found in this species. This finding suggests that positive selection swept one lysin allele and one 18-kDa allele to fixation. Similarities between mtCOI and lysin gene trees indicate that rapid adaptive evolution of lysin has occurred consistently through the history of the group. Comparisons with mtCOI molecular clock calibrations suggest that nonsynonymous substitutions accumulate 2–50 times faster in lysin and 18-kDa genes than in rapidly evolving mammalian genes.

Multiple independent origins of mitochondrial gene order in birds

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced OL (origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.

Evaluating multiple alternative hypotheses for the origin of Bilateria: An analysis of 18S rRNA molecular evidence

Six alternative hypotheses for the phylogenetic origin of Bilateria are evaluated by using complete 18S rRNA gene sequences for 52 taxa. These data suggest that there is little support for three of these hypotheses. Bilateria is not likely to be the sister group of Radiata or Ctenophora, nor is it likely that Bilateria gave rise to Cnidaria or Ctenophora. Instead, these data reveal a close relationship between bilaterians, placozoans, and cnidarians. From this, several inferences can be drawn. Morphological features that previously have been identified as synapomorphies of Bilateria and Ctenophora, e.g., mesoderm, more likely evolved independently in each clade. The endomesodermal muscles of bilaterians may be homologous to the endodermal muscles of cnidarians, implying that the original bilaterian mesodermal muscles were myoepithelial. Placozoans should have a gastrulation stage during development. Of the three hypotheses that cannot be falsified with the 18S rRNA data, one is most strongly supported. This hypothesis states that Bilateria and Placozoa share a more recent common ancestor than either does to Cnidaria. If true, the simplicity of placozoan body architecture is secondarily derived from a more complex ancestor. This simplification may have occurred in association with a planula-type larva becoming reproductive before metamorphosis. If this simplification took place during the common history that placozoans share with bilaterians, then placozoan genes that contain a homeobox, such as Trox2, should be explored, for they may include the gene or genes most closely related to Hox genes of bilaterians.

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.