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PubMed2004

A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac.

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.

Animals↗

PubMed1999

New technologies and DNA resources for high throughput biology.

The rapid increase in DNA sequencing information is opening up new opportunities in genetics. The current methods for processing and analysing genetic data are, however, slow and labour intensive. The next wave of genetic analysis will rely on the analysis of DNA variation from large population based cohorts. These studies will provide important new data on population and disease genetics and have the potential to make a significant impact on our current healthcare practices. In order for these studies to deliver, we need to develop a new generation of ultra-rapid DNA technologies which will allow us to generate, capture and efficiently exploit these new data. This chapter describes the recent advances in DNA sequencing and genotyping technologies that will lead to 100-1000-fold increases in our ability to produce the DNA data we need to explore and exploit the new genetic opportunities to the full.

DNA Mutational Analysis↗

PubMed2003

Two kinds of sex ratio distorters in a moth, Ostrinia scapulalis.

We surveyed female-biased sex ratio traits (SR traits) in a moth, Ostrinia scapulalis (Lepidoptera: Crambidae) from six geographic locations in Japan. A small proportion (8.3%) of females showed the SR trait. About half of them were associated with a Wolbachia infection (SRw+), whereas the remaining ones were not (SRw-). No significant differences in frequencies were detected among locations and years for either SRw+ or SRw- females. In SRw+ matrilines, the SR trait was inherited. Curing the Wolbachia infection by tetracycline at the larval stages resulted in all-male broods in the next generation. These characteristics were identical to the Wolbachia-induced SR trait observed previously in the related species Ostrinia furnacalis. The Wolbachia strain in O. scapulalis was indistinguishable from that in O. furnacalis in terms of the wsp and ftsZ gene sequences, suggesting either that infection originated from a common ancestor of the two hosts or that Wolbachia was transmitted horizontally between the host species. In SRw- matrilines, on the other hand, the SR trait was not due to bacterial infection but some other maternally inherited element, since antibiotic treatment had no effect and bacterial PCR did not give the characteristic product. In these matrilines, the SR trait occasionally failed to show, suggesting the presence of nuclear factor(s) suppressing the SR trait.

Animals↗

PubMed2003

Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites.

Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.

ADAM Proteins↗

PubMed2006

Poxvirus tumor necrosis factor receptor (TNFR)-like T2 proteins contain a conserved preligand assembly domain that inhibits cellular TNFR1-induced cell death.

The poxvirus tumor necrosis factor receptor (TNFR) homologue T2 has immunomodulatory properties; secreted myxoma virus T2 (M-T2) protein binds and inhibits rabbit TNF-alpha, while intracellular M-T2 blocks virus-induced lymphocyte apoptosis. Here, we define the antiapoptotic function as inhibition of TNFR-mediated death via a highly conserved viral preligand assembly domain (vPLAD). Jurkat cell lines constitutively expressing M-T2 were generated and shown to be resistant to UV irradiation-, etoposide-, and cycloheximide-induced death. These cells were also resistant to human TNF-alpha, but M-T2 expression did not alter surface expression levels of TNFRs. Previous studies indicated that T2's antiapoptotic function was conferred by the N-terminal region of the protein, and further examination of this region revealed a highly conserved N-terminal vPLAD, which is present in all poxvirus T2-like molecules. In cellular TNFRs and TNF-alpha-related apoptosis-inducing ligand (TRAIL) receptors (TRAILRs), PLAD controls receptor signaling competency prior to ligand binding. Here, we show that M-T2 potently inhibits TNFR1-induced death in a manner requiring the M-T2 vPLAD. Furthermore, we demonstrate that M-T2 physically associates with and colocalizes with human TNFRs but does not prevent human TNF-alpha binding to cellular receptors. Thus, M-T2 vPLAD is a species-nonspecific dominant-negative inhibitor of cellular TNFR1 function. Given that the PLAD is conserved in all known poxvirus T2-like molecules, we predict that it plays an important function in each of these proteins. Moreover, that the vPLAD confers an important antiapoptotic function confirms this domain as a potential target in the development of the next generation of TNF-alpha/TNFR therapeutics.

Amino Acid Sequence↗

PubMed1993

Multigenerational amplification of a reference ladder for alleles at locus D1S80.

The interlaboratory typing of DNA specimens that have been amplified at the D1S80 locus necessitates the use of a standard allelic reference ladder. This communication describes a technique in which individual, amplified alleles are isolated, combined, and amplified by PCR to produce a functional reference ladder composed of many of the alleles that occur at this locus. The amplified ladder can serve directly as a template source for production of the next generation of reference ladder. This process, in which each amplified ladder serves as the template for the next has been carried through multiple generations.

Alleles↗

PubMed1997

Expression plasmid vectors with convenient subcloning sites in lambda gt11 that efficiently produce detectable tagged proteins.

We have generated cDNA expression vectors that efficiently produce tagged proteins. The newly introduced cloning site of this plasmid facilitates subcloning of cDNA in the lambda gt11 phage into the plasmid vector. Because the cDNA is inserted next to the motifs of the tagged DNA sequence, the protein produced by the tag sequence-coupled cDNA is easily detected by Western blot analysis or immunoprecipitation using commercially available antibodies. The double-tagged protein significantly enhances the efficiency of Western blot and immunoprecipitation detection as compared with the single-tagged protein.

3T3 Cells↗

PubMed1990

Effect of endotoxin-induced cell injury on 70-kD heat shock proteins in bovine lung endothelial cells.

Heat shock proteins (HSPs) have been remarkably conserved throughout evolution. It has been assumed that induction of HSPs remains a stereotypic response to injury, important for survival of eukaryotic cells during euthermic injury. However, there are few studies of this phenomenon in endothelial cells, and none in pulmonary endothelial cells. We studied the induction of synthesis of 70-kD proteins in bovine pulmonary artery endothelial cells (BPAECs) in response to heat shock and to euthermic injury induced by bacterial endotoxin. First, in response to heat, BPAECs showed rapid and reversible heat-induced synthesis of 70-kD proteins, readily detectable by one-dimensional SDS-PAGE of [35S]methionine-labeled BPAECs. Heat shock at 42 degrees C for 3 h or 43 degrees C for 2 h suppressed total protein synthesis by 30% (P less than 0.001) but an increased rate of synthesis of 70-kD protein continued, representing an increasing fraction of total protein synthesis. Heat-induced synthesis of 70-kD protein returned to baseline levels 8 h after heat shock. Northern analysis showed that mRNA for a protein homologous to a conserved amino acid sequence in the family of species-homologous 70-kD heat shock proteins (HSP 70) was induced by a 15-min incubation at 42 degrees C and remained detectably increased for 6 h. We next assessed whether euthermic injury by bacterial endotoxin (LPS) generated a similar response. LPS was cytotoxic by BPAECs as assessed morphologically, by release of 51Cr from prelabeled cells, and by a significant suppression of total protein synthesis (range, 35 to 70%; P less than 0.001). Despite cytotoxicity, LPS did not induce 70-kD protein at a level that could be detected by SDS-PAGE, and no increase in mRNA for HSP 70 was detected by Northern analysis. LPS-injured BPAECs remained "competent" to induce both 70-kD proteins and mRNA for HSP 70 in response to heat shock. We conclude that at least quantitatively, induction of HSP 70 by BPAECs is not a stereotypic response to injury but rather is at least relatively injury-specific. However, competence to induce HSP 70 appears to be extremely resilient: it is retained in dysfunctional BPAECs in the face of profound inhibition of global protein synthesis, suggesting an important homeostatic role.

Animals↗

PubMed2006

Gene expression profiling of chicken primordial germ cell ESTs.

BACKGROUND: Germ cells are the only cell type that can penetrate from one generation to next generation. At the early embryonic developmental stages, germ cells originally stem from primordial germ cells, and finally differentiate into functional gametes, sperm in male or oocyte in female, after sexual maturity. This study was conducted to investigate a large-scale expressed sequence tag (EST) analysis in chicken PGCs and compare the expression of the PGC ESTs with that of embryonic gonad. RESULTS: We constructed 10,851 ESTs from a chicken cDNA library of a collection of highly separated embryonic PGCs. After chimeric and problematic sequences were filtered out using the chicken genomic sequences, there were 5,093 resulting unique sequences consisting of 156 contigs and 4,937 singlets. Pearson chi-square tests of gene ontology terms in the 2nd level between PGC and embryonic gonad set showed no significance. However, digital gene expression profiling using the Audic's test showed that there were 2 genes expressed significantly with higher number of transcripts in PGCs compared with the embryonic gonads set. On the other hand, 17 genes in embryonic gonads were up-regulated higher than those in the PGC set. CONCLUSION: Our results in this study contribute to knowledge of mining novel transcripts and genes involved in germline cell proliferation and differentiation at the early embryonic stages.

Animals↗

PubMed2005

The Baltic Health Network - Taking Secure, Internet-based Healthcare Networks to the Next Level.

Internet-based health care networks are a step forward compared to first generation health care networks, which has been limited to pushing text-based messages between different systems. An Internet-based network can also "pull" data - and not only text but any digital data - for instance images and video sequences. The Internet-based networks can more effectively fulfil the vision of access to relevant data regardless of time and location. Although far from identical, the health delivery systems of Denmark, Norway and Sweden are similar. They also share a shortage of specialized health personnel - not least radiologists and in some regions obstetricians. Furthermore, over the past ten years they have implemented an IT-strategy to increase efficiency in the delivery of healthcare services. Part of this strategy has been to build three national networks on top of the existing regional, secure and Internet-based healthcare networks. These national networks connect not only all hospitals in the three countries, but also a majority of the other stakeholders in the healthcare sector (GPs, private specialists, laboratories, homecare services etc.). The organizations behind the three networks are now working on creating a trans-national network, the Baltic Health Network (BHN), which will be one of the outcomes of the Baltic eHealth project and will not only connect the three national networks but also add two hospital networks from Lithuania and Estonia. The BHN is expected to be operational by June 2005. One of major advantages of the BHN is that the many rural hospitals of the Baltic Sea Area with a few mouse clicks can reach a specialist for second opinion in any of the approximately 200 hospitals connected to the network. For instance the midwives in the rural areas of Västerbottan County, Sweden, are awaiting the establishment of BHN to get access to second opinions from specialists at National Center for Foetal Medicine at the University Hospital of Trondheim, Norway. The BHN will remove a very important technical barrier for collaboration between health professionals and the Baltic eHealth project hopes that this and other project initiatives will facilitate the large-scale usage of second opinion from available health care experts regardless of institutional, regional and even national borders. This will lift the quality of service to patients in the Baltic Sea Region - especially in the rural areas where highly specialized health professionals tend to be geographically far away.

Cooperative Behavior↗

PubMed2001

Structural basis of caspase-7 inhibition by XIAP.

The inhibitor of apoptosis (IAP) proteins suppress cell death by inhibiting the catalytic activity of caspases. Here we present the crystal structure of caspase-7 in complex with a potent inhibitory fragment from XIAP at 2.45 A resolution. An 18-residue XIAP peptide binds the catalytic groove of caspase-7, making extensive contacts to the residues that are essential for its catalytic activity. Strikingly, despite a reversal of relative orientation, a subset of interactions between caspase-7 and XIAP closely resemble those between caspase-7 and its tetrapeptide inhibitor DEVD-CHO. Our biochemical and structural analyses reveal that the BIR domains are dispensable for the inhibition of caspase-3 and -7. This study provides a structural basis for the design of the next-generation caspase inhibitors.

Apoptosis↗

PubMed1987

An approach to the multiple-minima problem in protein folding by relaxing dimensionality. Tests on enkephalin.

An algorithm for locating the region in conformational space containing the global energy minimum of a polypeptide is described. Distances are used as the primary variables in the minimization of an objective function that incorporates both energetic and distance-geometric terms. The latter are obtained from geometry and energy functions, rather than nuclear magnetic resonance experiments, although the algorithm can incorporate distances from nuclear magnetic resonance data if desired. The polypeptide is generated originally in a space of high dimensionality. This has two important consequences. First, all interatomic distances are initially at their energetically most favorable values; i.e. the polypeptide is initially at a global minimum-energy conformation, albeit a high-dimensional one. Second, the relaxation of dimensionality constraints in the early stages of the minimization removes many potential energy barriers that exist in three dimensions, thereby allowing a means of escaping from three-dimensional local minima. These features are used in an algorithm that produces short trajectories of three-dimensional minimum-energy conformations. A conformation in the trajectory is generated by allowing the previous conformation in the trajectory to evolve in a high-dimensional space before returning to three dimensions. The resulting three-dimensional structure is taken to be the next conformation in the trajectory, and the process is iterated. This sequence of conformations results in a limited but efficient sampling of conformational space. Results for test calculations on Met-enkephalin, a pentapeptide with the amino acid sequence H-Tyr-Gly-Gly-Phe-Met-OH, are presented. A tight cluster of conformations (in three-dimensional space) is found with ECEPP energies (Empirical Conformational Energy Program for Peptides) lower than any previously reported. This cluster of conformations defines a region in conformational space in which the global-minimum-energy conformation of enkephalin appears to lie.

Algorithms↗

PubMed2000

Genetic regulation of somite formation.

Segmentation of the paraxial mesoderm into somites requires a strategy distinct from the division of a preexisting field of cells, as seen in the segmentation of the vertebrate hindbrain into rhombomeres and the formation of the body plan of invertebrates. Each new somite forms from the anterior end of the segmental plate; therefore, the conditions for establishing the anterior-posterior boundary must be re-created prior to the formation of the next somite. It has been established that regulation of this process is native to the anterior end of the segmental plate, however, the components of a genetic pathway are poorly understood. A growing library of candidate genes has been generated from hybridization screens and sequence homology searches, which include cell adhesion molecules, cell surface receptors, growth factors, and transcription factors. With the increasing accessibility of gene knockout technology, many of these genes have been tested for their role in regulating somitogenesis. In this chapter, we will review the significant advances in our understanding of segmentation based on these experiments.

Animals↗

PubMed2000

Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues.

In DNA, the deamination of dAMP generates 2'-deoxy-inosine 5'-monophosphate (dIMP). Hypoxanthine (HX) residues are mutagenic since they give rise to A.T-->G.C transition. They are excised, although with different efficiencies, by an activity of the 3-methyl-adenine (3-meAde)-DNA glycosylases from Escherichia coli (AlkA protein), human cells (ANPG protein), rat cells (APDG protein) and yeast (MAG protein). Comparison of the kinetic constants for the excision of HX residues by the four enzymes shows that the E.coli and yeast enzymes are quite inefficient, whereas for the ANPG and the APDG proteins they repair the HX residues with an efficiency comparable to that of alkylated bases, which are believed to be the primary substrates of these DNA glycosylases. Since the use of various substrates to monitor the activity of HX-DNA glycosylases has generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases of different origin was compared using three different substrates. Moreover, using oligo-nucleotides containing a single dIMP residue, we investigated a putative sequence specificity of the enzymes involving the bases next to the HX residue. We found up to 2-5-fold difference in the rates of HX excision between the various sequences of the oligonucleotides studied. When the dIMP residue was placed opposite to each of the four bases, a preferential recognition of dI:T over dI:dG, dI:dC and dI:dA mismatches was observed for both human (ANPG) and E.coli (AlkA) proteins. At variance, the yeast MAG protein removed more efficiently HX from a dI:dG over dI:dC, dI:T and dI:dA mismatches.

Animals↗

PubMed2005

Plant eco-devo: the potential of poplar as a model organism.

Ecological developmental genetics is the study of how ecologically significant traits originate in the genome and how the allelic combinations responsible are maintained in populations and species. Plant development involves a continuous feedback between growth and environment and the success of individual genotype x environment interactions determines the passage of alleles to the next generation: the adaptive recursion. Outbreeding plants contain a large amount of genetic variation, mostly in the form of single nucleotide polymorphisms (SNPs). One of the challenges of eco-devo is to distinguish neutral SNPs from those with ecological consequences. The complete genome sequence of Populus trichocarpa Torr. & A. Gray will be a significant aid in this endeavour. Occurring from California to Alaska, this is the first ecologically 'keystone' species to be sequenced. It has a rich natural history and is an obligate outbreeder. The individual sequenced, Nisqually-1, appears to be heterozygous on average about every 100 bp over the c. 500 million bp of the genome. Overlaid on this within-individual variation is some ecologically based between-individual genotypic variation evident across the distribution of the species. The synthesis of information from genomics and ecology is now in prospect. This 'ecomolecular synthesis' is likely to provide a rich insight into the genomic basis of plant adaptation.

Adaptation, Physiological↗

PubMed2006

Vertical transmission of diverse microbes in the tropical sponge Corticium sp.

Sponges are host to extremely diverse bacterial communities, some of which appear to be spatiotemporally stable, though how these consistent associations are assembled and maintained from one sponge generation to the next is not well understood. Here we report that a diverse group of microbes, including both bacteria and archaea, is consistently present in aggregates within embryos of the tropical sponge Corticium sp. The major taxonomic groups represented in bacterial 16S rRNA sequences amplified from the embryos are similar to those previously described in a variety of marine sponges. Three selected bacterial taxa, representing proteobacteria, actinobacteria, and a clade including recently described sponge-associated bacteria, were tested and found to be present in all adult samples tested over a 3-year period and in the embryos throughout development. Specific probes were used in fluorescence in situ hybridization to localize cells of the three types in the embryos and mesohyl. This study confirms the vertical transmission of multiple, phylogenetically diverse microorganisms in a marine sponge, and our findings lay the foundation for future work on exploring vertical transmission of specific, yet diverse, microbial assemblages in marine sponges.

Animals↗

PubMed1984

Origins of replication and gene regulation.

Eukaryotic chromosomes appear to consist of many replicons, the time of replication of which is probably controlled by specific origins. However, plasmids without specific eukaryotic origins may also replicate in some cells when injected into nuclei or transferred during transformation. The efficiency and the mechanisms of their initiation are still uncertain. A number of reports are cited which indicate that natural eukaryotic DNAs initiate their replication from specific origins. The nature of these origins are known in only a few instances and no general conclusions can yet be given about the nucleotide sequences involved. Short dispersed repeats of the Alu type appear to function as origins since they enhance the efficiency of replication of vector plasmids in Xenopus eggs. Certain sequences from a variety of eukaryotic DNAs also enhance the replicative potential of plasmids in yeast cells. The common features of such initiators or enhancers is uncertain. If dispersed repeats are origins in mammalian chromosomes, the number appears to be excessive. Either only a subset are functional, or the functional ones are only suborigins in larger replicons in which master origins (not yet isolated) function in the regulation of the timing of replication. Evidence is cited which indicates that the regulation of the time of replication of a gene or gene cluster is part of a regulatory system that makes the DNA available for transcription or leaves it in an inactive state. About one-half the DNA in mammalian cells is replicated in the first half of S phase (SE). After a brief pause in mid-S phase, the remainder of the DNA is replicated in what is designated late S (SL). The fractions replicated in SE and SL may vary in other phylogenetic groups, but wherever division of differentiated cells occurs such fractions are likely to be found. The following hypothesis is proposed. The DNA replicated in SL is suppressed in transcription, if it has the appropriate promoter regions, because the newly replicated DNA is complexed with proteins that suppress transcription. These proteins are only available during SL. Those genes replicated in SE are complexed with a different set of proteins which leave the promoter regions open for transcription when the appropriate regulatory molecules are available. In this way an inactive state or potentially active state can be transmitted from one cell generation to the next. Evidence is cited which indicates that genes which are active in all cells at some stage in the cell cycle are replicated in SE.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2000

Spermatocyte-specific gene excision by targeted expression of Cre recombinase.

Transgenic mice carrying the coding sequence of the Cre recombinase, whose expression was driven by the spermatocyte-specific Pgk-2 promoter, were generated. These mice were crossed with a reporter transgenic line, which produces beta-galactosidase depending on the occurrence of loxP-mediated DNA recombination. When DNA of the offspring was analyzed by PCR and Southern blotting, signals that appear after the recombination were detectable only in the testis. Histochemical analyses revealed that beta-galactosidase was present in spermatocytes and spermatogenic cells at later differentiation stages. However, the distribution of the protein was not uniform in all spermatocytes. Analyses of genomic DNA of the next generation indicated that recombination took place in about 70% of spermatogenic cells. From these results, we concluded that this transgenic line possessing Pgk-2-driven expression of the Cre recombinase should be useful for identifying spermatogenic genes that function at or after the spermatocyte stage.

Animals↗