Showing papers by "Laboratory of Molecular Biology published in 2004"

Global Mapping of the Yeast Genetic Interaction Network

Abstract: A genetic interaction network containing approximately 1000 genes and approximately 4000 interactions was mapped by crossing mutations in 132 different query genes into a set of approximately 4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects. Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway. The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions. Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.

BAR domains as sensors of membrane curvature: the amphiphysin BAR structure

Abstract: The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

Regulatory T cells mediate maternal tolerance to the fetus.

Abstract: Pregnancy constitutes a major challenge to the maternal immune system, as it has to tolerate the persistence of paternal alloantigen Although localized mechanisms contribute to fetal evasion from immune attack, maternal alloreactive lymphocytes persist We demonstrate here an alloantigen-independent, systemic expansion of the maternal CD25+ T cell pool during pregnancy and show that this population contains dominant regulatory T cell activity In addition to their function in suppressing autoimmune responses, maternal regulatory T cells suppressed an aggressive allogeneic response directed against the fetus Their absence led to a failure of gestation due to immunological rejection of the fetus

The dynamin superfamily: universal membrane tubulation and fission molecules?

Abstract: Dynamins are large GTPases that belong to a protein superfamily that, in eukaryotic cells, includes classical dynamins, dynamin-like proteins, OPA1, Mx proteins, mitofusins and guanylate-binding proteins/atlastins. They are involved in many processes including budding of transport vesicles, division of organelles, cytokinesis and pathogen resistance. With sequenced genomes from Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, yeast species and Arabidopsis thaliana, we now have a complete picture of the members of the dynamin superfamily from different organisms. Here, we review the superfamily of dynamins and their related proteins, and propose that a common mechanism leading to membrane tubulation and/or fission could encompass their many varied functions.

Genomic analysis of regulatory network dynamics reveals large topological changes

Abstract: Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids. It has recently been used in molecular biology, but so far the resulting networks have only been analysed statically. Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae. We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics. We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations. In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network. A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions. By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors). Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy. We anticipate that many of the concepts presented here--particularly the large-scale topological changes and hub transience--will apply to other biological networks, including complex sub-systems in higher eukaryotes.

Complete genome sequence of the apicomplexan, Cryptosporidium parvum.

Abstract: The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.

GSK3 inhibitors: development and therapeutic potential

Abstract: Glycogen synthase kinase-3 (GSK3) was initially identified more than two decades ago as an enzyme involved in the control of glycogen metabolism. In recent years it has been shown to have key roles in regulating a diverse range of cellular functions, which have prompted efforts to develop GSK3 inhibitors as therapeutics. Here, we describe the biology of GSK3 relevant to its potential as a target for diabetes and neurodegenerative diseases, and discuss progress in the development of GSK3 inhibitors.

A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses.

Abstract: Legumes can enter into symbiotic relationships with both nitrogen-fixing bacteria (rhizobia) and mycorrhizal fungi. Nodulation by rhizobia results from a signal transduction pathway induced in legume roots by rhizobial Nod factors. DMI3, a Medicago truncatula gene that acts immediately downstream of calcium spiking in this signaling pathway and is required for both nodulation and mycorrhizal infection, has high sequence similarity to genes encoding calcium and calmodulin-dependent protein kinases (CCaMKs). This indicates that calcium spiking is likely an essential component of the signaling cascade leading to nodule development and mycorrhizal infection, and sheds light on the biological role of plant CCaMKs.

High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma.

Abstract: In a recent issue of Genes, Chromosomes & Cancer, van den Berg et al. (2003) reported on the high expression of human BIC RNA in Hodgkin lymphoma by means of the serial analysis of gene expression (SAGE) technique. In addition, using a combination of RNA in situ hybridization and immunostaining, they found that the expression of BIC is specific for Reed–Sternberg (RS) cells and that the BIC transcripts were in the nuclei of the RS cells. In contrast to their findings in Hodgkin lymphoma, an analysis of 43 cases of non-Hodgkin lymphoma (NHL; 15 follicular lymphomas, 7 diffuse large B-cell lymphomas, 9 Burkitt lymphomas, 7 anaplastic large cell lymphomas, and 5 T-cell-rich B-cell lymphomas) did not reveal any remarkable up-regulation of BIC expression, although one case of Burkitt lymphoma (BL) showed weak expression of BIC in a minority of its tumor cells. The BIC locus was originally identified as a common retroviral integration site in avian-leukosis virus–induced B–cell lymphomas (Clurman and Hayward, 1989; Tam et al., 1997). It should be stressed that the human BIC gene encodes a microRNA, miR-155. This microRNA is encoded by nucleotides 241–262 of BIC, which spans 1,421 bp in total and is on chromosome 21 (GenBank accession number: AF402776). MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that interact with coding mRNA and trigger either translation repression or direct RNA cleavage via RNA interference, depending on the degree of complementarity with the specific target mRNA (Ambros, 2001; Lagos-Quintana et al., 2001; Ruvkun, 2001; Lai, 2002; Pasquinelli, 2002; Ambros et al., 2003; Lagos-Quintana et al., 2003). Mature miRNAs are 21–23 nt long and are excised from an approximately 60to 80-nt double-stranded RNA hairpin by Dicer RNase III (Hutvagner et al., 2001). In the last 2 years, more than 200 human microRNA genes have been described, but the prediction and validation of their target mRNAs by computerized means and experimental approaches is a tantalizing and still unresolved task (Ambros et al., 2003). Apart from this open question, it was recently hypothesized that microRNA genes might play an important role in oncogenesis (McManus, 2003). We support the idea advanced by van den Berg et al. that BIC might play a role in the selection of B cells, but here we also extend their data by demonstrating that miR-155/BIC is highly expressed in pediatric BL but not in other hematologic malignancies, for example, pre-B/common or T-cell leukemia. The conditio sine qua non for the development of BL is activation of the MYC oncogene, mostly by chromosomal translocations in which MYC is juxtaposed to an immunoglobulin enhancer. On the other hand, there is a body of evidence that the activation of MYC alone is not sufficient for full malignancy. Providing support for this contention was the demonstration that MYC cooperates with BMI1, PIM1, RAF, BCL2, or, as shown in a very recent report, with the Werner syndrome protein WRN (Grandori et al., 2003) We analyzed tumor cells from 21 children (ages 2–13 years, with a median age of 6 years) with BL (n 11), common/pre-B acute lymphoblastic leukemia (ALL; n 6), or T-cell ALL (n 4). In all cases selected, the proportion of tumor cells was 80% or greater. The presence of an IGH/MYC rearrangement was demonstrated by long-distance polymerase chain reaction (PCR) in all BL cases, whereas neither the common/pre-B nor T-cell ALLs had such a recombination (Busch et al., unpublished data). All patients were treated according to the NHL-BFM 90, 95, or the ALL-BFM 90, 95 multicenter therapy study (Reiter et al., 1999; Schrappe et al., 2000). Immunophenotyping was done according to standard procedures (Ludwig et al., 1994). Informed parental consent was obtained in all cases. RNA isolated from peripheral blood from 11 healthy volunteers served as an additional control.

The Small RNA Regulators of Escherichia coli: Roles and Mechanisms*

Abstract: Small noncoding RNAs have been found in all organisms, primarily as regulators of translation and message stability. The most exhaustive searches have taken place in E. coli, resulting in identification of more than 50 small RNAs, or 1%-2% of the number of protein-coding genes. One large class of these small RNAs uses the RNA chaperone Hfq; members of this class act by pairing to target messenger RNAs. Among the members of this class are DsrA and RprA, which positively regulate rpoS translation, OxyS, which negatively regulates rpoS translation and fhlA translation, RyhB, which reapportions iron use in the cell by downregulating translation of many genes that encode Fe-containing proteins, and Spot 42, which changes the polarity of translation in the gal operon. The promoters of these small RNAs are tightly regulated, frequently as part of well-understood regulons. Lessons learned from the study of small RNAs in E. coli can be applied to finding these important regulators in other organisms.

ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance.

Abstract: The Toll-interleukin 1 receptor (TIR) superfamily, defined by the presence of an intracellular TIR domain, initiates innate immunity through activation of the transcription factor NF-kappa B, leading to the production of proinflammatory cytokines. ST2 is a member of the TIR family that does not activate NF-kappa B and has been suggested as an important effector molecule of T helper type 2 (T(H)2) responses. We show here that the membrane-bound form of ST2 negatively regulated type I interleukin 1 receptor (IL-1RI) and Toll-like receptor 4 (TLR4) but not TLR3 signaling by sequestrating the adaptors MyD88 and Mal. In contrast to wild-type mice, ST2-deficient mice failed to develop endotoxin tolerance. Thus, these results provide a molecular explanation for the function of ST2 in T(H)2 responses, as inhibition of TLRs promotes a T(H)2 response, and also identify ST2 as a key regulator of endotoxin tolerance.

Gene regulatory network growth by duplication

Abstract: We are beginning to elucidate transcriptional regulatory networks on a large scale1 and to understand some of the structural principles of these networks2,3, but the evolutionary mechanisms that form these networks are still mostly unknown. Here we investigate the role of gene duplication in network evolution. Gene duplication is the driving force for creating new genes in genomes: at least 50% of prokaryotic genes4,5 and over 90% of eukaryotic genes6 are products of gene duplication. The transcriptional interactions in regulatory networks consist of multiple components, and duplication processes that generate new interactions would need to be more complex. We define possible duplication scenarios and show that they formed the regulatory networks of the prokaryote Escherichia coli and the eukaryote Saccharomyces cerevisiae. Gene duplication has had a key role in network evolution: more than one-third of known regulatory interactions were inherited from the ancestral transcription factor or target gene after duplication, and roughly one-half of the interactions were gained during divergence after duplication. In addition, we conclude that evolution has been incremental, rather than making entire regulatory circuits or motifs by duplication with inheritance of interactions.

Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators

Abstract: Patients presenting with metastatic rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children, have a very poor clinical prognosis. This is due, in large part, to our rudimentary knowledge of the molecular events that dictate metastatic potential. We used cDNA microarray analysis of RMS cell lines, derived from Ink4a/Arf-deficient mice transgenic for hepatocyte growth factor/scatter factor (HGF/SF), to identify a set of genes whose expression was significantly different between highly and poorly metastatic cells. Subsequent in vivo functional studies revealed that the actin filament-plasma membrane linker ezrin (encoded by Vil2) and the homeodomain-containing transcription factor Six-1 (sine oculis-related homeobox-1 homolog) had essential roles in determining the metastatic fate of RMS cells. VIL2 and SIX1 expression was enhanced in human RMS tissue, significantly correlating with clinical stage. The identification of ezrin and Six-1 as critical regulators of metastasis in RMS provides new mechanistic and therapeutic insights into this pediatric cancer.

The genome of Cryptosporidium hominis

Abstract: Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa--protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome approximately 9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

The macro domain is an ADP-ribose binding module

Abstract: The ADP-ribosylation of proteins is an important post-translational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long-term memory formation. Yet no protein modules are known that specifically recognize the ADP-ribose nucleotide. We provide biochemical and structural evidence that macro domains are high-affinity ADP-ribose binding modules. Our structural analysis reveals a conserved ligand binding pocket among the macro domain fold. Consistently, distinct human macro domains retain their ability to bind ADP-ribose. In addition, some macro domain proteins also recognize poly-ADP-ribose as a ligand. Our data suggest an important role for proteins containing macro domains in the biology of ADP-ribose.

Mesothelin: A new target for immunotherapy

Abstract: Mesothelin is a differentiation antigen present on normal mesothelial cells and overexpressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma. The mesothelin gene encodes a precursor protein that is processed to yield the 40-kDa protein, mesothelin, attached to the cell membrane by a glycosylphosphatidyl inositol linkage and a 31-kDa shed fragment named megakaryocyte-potentiating factor. The biological function of mesothelin is not known. Mesothelin is a promising candidate for tumor-specific therapy, given its limited expression in normal tissues and high expression in several cancers. SS1(dsFv)PE38 is a recombinant anti-mesothelin immunotoxin that is undergoing clinical evaluation in patients with mesothelin-expressing tumors. There is evidence that mesothelin is an immunogenic protein and could be exploited as a therapeutic cancer vaccine. A soluble mesothelin variant has been identified and could be a useful tumor marker for malignant mesotheliomas.

Dynamics of putative raft-associated proteins at the cell surface.

Abstract: Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (>4 μm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis.

Abstract: Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.

Sparse-graph codes for quantum error correction

Abstract: Sparse-graph codes appropriate for use in quantum error-correction are presented. Quantum error-correcting codes based on sparse graphs are of interest for three reasons. First, the best codes currently known for classical channels are based on sparse graphs. Second, sparse-graph codes keep the number of quantum interactions associated with the quantum error-correction process small: a constant number per quantum bit, independent of the block length. Third, sparse-graph codes often offer great flexibility with respect to block length and rate. We believe some of the codes we present are unsurpassed by previously published quantum error-correcting codes.

Involvement of a novel transcriptional activator and small RNA in post‐transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system

Abstract: RyaA is a small non-coding RNA in Escherichia coli that was identified by its ability to bind tightly to the RNA chaperone Hfq. This study reports the role of RyaA in mediating the cellular response to glucose-specific phosphoenolypyruvate phosphotransferase system (PTS)-dependent phosphosugar stress. Aiba and co-workers have shown that a block in the metabolism of glucose 6-phosphate causes transient growth inhibition and post-transcriptional regulation of ptsG, encoding the glucose-specific PTS transporter. We found that RyaA synthesis was induced by a non-metabolizable glucose phosphate analogue and was necessary for relief of the toxicity of glucose phosphate stress. Expression of RyaA was sufficient to cause a rapid loss of ptsG mRNA, probably reflecting degradation of the message mediated by RyaA:ptsG pairing. The ryaA gene was renamed sgrS, for sugar transport-related sRNA. Expression of sgrS is regulated by a novel transcriptional activator, SgrR (formerly YabN), which has a putative DNA-binding domain and a solute-binding domain similar to those found in certain transport proteins. Our results suggest that under conditions of glucose phosphate accumulation, SgrR activates SgrS synthesis, causing degradation of ptsG mRNA. Decreased ptsG mRNA results in decreased production of glucose transport machinery, thus limiting further accumulation of glucose phosphate.

Early impairment of endothelial structure and function in young normal-weight women with polycystic ovary syndrome.

Abstract: The aim of this study was to evaluate the presence of early vascular damage in young normal-weight women with polycystic ovary syndrome (PCOS). Thirty young normal-weight women with PCOS, who had no additional metabolic or cardiovascular diseases, and 30 healthy women (controls) matched for age and body mass index were studied. A complete hormonal assay was performed in each subject. Serum insulin and glucose levels were measured at baseline and after the oral glucose tolerance test. Plasma endothelin-1 levels and serum lipid profile were also assessed. The endothelial function was studied by flow-mediated dilation on the brachial artery, and arterial structure was evaluated by intima-media thickness measurement using Doppler ultrasound of both common carotid arteries.A significant (P < 0.05) difference in flow-mediated dilation (14.3 +/- 1.9% vs. 18.1 +/- 2.0% for PCOS patients and controls, respectively) and in intima-media thickness (0.53 +/- 0.09 mm vs. 0.39 +/- 0.08 mm for PCOS patients and controls, respectively) was found between PCOS and control subjects. Serum endothelin-1 levels were also significantly (P < 0.05) higher in PCOS patients compared with controls (1.1 +/- 0.4 pmol/liter vs. 0.5 +/- 0.2 pmol/liter for PCOS patients and controls, respectively).In conclusion, our data show that young, normal-weight, nondyslipidemic, nonhypertensive women with PCOS have an early impairment of endothelial structure and function.

The role of modifications in codon discrimination by tRNA Lys UUU

Abstract: The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNALysUUU requires the modification N6-threonylcarbamoyladenosine at position 37 (t6A37), adjacent and 3′ to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t6A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm5U34 or s2U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.