Showing papers by "Laboratory of Molecular Biology published in 1995"

Transcription and translation.

Abstract: Publisher Summary This chapter reviews the current state of the knowledge of transcription and translation in Caenorhabditis elegans. Transcription in eukaryotes is accomplished by three RNA polymerases, each transcribing a particular type of RNA; RNA polymerase I transcribes ribosomal RNA, RNA polymerase II transcribes mRNAs and snRNAs, and RNA polymerase III transcribes tRNAs and other low-molecular-weight RNAs. The chapter focuses on RNA polymerase II transcription. The C. elegans gene for the large subunit of RNA polymerase II has been cloned, and has shown to encode a protein that resembles its vertebrate counterparts. For most polymerase II transcribed genes in C. elegans, a sequence fitting the consensus TATA element about 30 bp upstream of the transcriptional start site is found. Recently, a cDNA encoding the TATA box binding protein, or TATA binding protein (TBP) subunit of transcription factor II D (TFIID), has been identified in the nematode. This protein shows extended regions of sequence similarity to vertebrate TBP, binds to a TATA box sequence and can substitute for vertebrate TFIID basal activity in in vitro extracts. These results illustrate a high degree of evolutionary conservation in the basic transcription machinery.

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.

Abstract: Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associated energy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of approximately 10(5) molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10,000 particles are required. Below 10(5) molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

Acetylcholine receptor channel imaged in the open state

Abstract: The structure of the open-channel form of the acetylcholine receptor has been determined from electron images of Torpedo ray postsynaptic membranes activated by brief (< 5 ms) mixing with droplets containing acetylcholine. Comparison with the closed-channel form shows that acetylcholine initiates small rotations of the subunits in the extracellular domain, which trigger a change in configuration of alpha-helices lining the membrane-spanning pore. The open pore tapers towards the intracellular membrane face, where it is shaped by a 'barrel' of alpha-helices having a pronounced right-handed twist.

E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers.

Abstract: Compelling experimental evidence exists for a potent invasion suppressor role of the cell-cell adhesion molecule E-cadherin. In addition, a tumour suppressor effect has been suggested for E-cadherin. In human cancers, partial or complete loss of E-cadherin expression correlates with malignancy. To investigate the molecular basis for this altered expression we developed a comprehensive PCR/SSCP mutation screen for the human E-cadherin gene. For 49 breast cancer patients the occurrence of tumour-specific mutations in the E-cadherin gene was examined. No relevant DNA changes were encountered in any of 42 infiltrative ductal or medullary breast carcinoma samples. In contrast, four out of seven infiltrative lobular breast carcinomas harboured protein truncation mutations (three nonsense and one frameshift) in the extracellular part of the E-cadherin protein. Each of the four lobular carcinomas with E-cadherin mutations showed tumour-specific loss of heterozygosity of chromosomal region 16q22.1 containing the E-cadherin locus. In compliance with this, no E-cadherin expression was detectable by immunohistochemistry in these four tumours. These findings offer a molecular explanation for the typical scattered tumour cell growth in infiltrative lobular breast cancer.

Protein motifs 5. Zinc fingers.

Abstract: The term zinc finger was first used to describe a 30-residue, repeated sequence motif found in an unusually abundant Xenopus transcription factor. It was proposed that each motif is folded around a central zinc ion to form an independent minidomain and that adjacent zinc fingers are combined as modules to make up a DNA-binding domain with the modules "gripping" the DNA (hence the term finger). We now know that these proposals were correct and that these DNA-binding motifs are found in many eukaryotic DNA-binding proteins. More recently, crystal structures of three different complexes between zinc finger domains and their target DNA binding sites have revealed a remarkably simple mode of interaction with DNA. The simplicity of the zinc finger structure, and of its interaction with DNA, is a very striking feature of this protein domain. After the discovery of the zinc finger motif, patterns of potential zinc ligands have been found in several other proteins, some of which also bind to DNA. Structural studies of these domains have revealed how zinc can stabilize quite diverse protein architectures. In total, 10 such small zinc-binding domains have been studied structurally. These form a diverse collection, but each in turn has been termed a zinc finger motif-although clearly what they have in common is only their zinc-binding property, which stabilizes an apparently autonomously folded unit.

Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.

Abstract: Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.

The 8.5 A projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits.

Abstract: Two-dimensional crystals from light-harvesting complex I (LHC I) of the purple non-sulfur bacterium Rhodospirillum rubrum have been reconstituted from detergent-solubilized protein complexes. Frozen-hydrated samples have been analysed by electron microscopy. The crystals diffract beyond 8 A and a projection map was calculated to 8.5 A. The projection map shows 16 subunits in a 116 A diameter ring with a 68 A hole in the centre. These dimensions are sufficient to incorporate a reaction centre in vivo. Within each subunit, density for the alpha- and the beta-polypeptide chains is clearly resolved, and the density for the bacteriochlorophylls can be assigned. The experimentally determined structure contradicts models of the LHC I presented so far.

Overexpression of integral membrane proteins for structural studies

Abstract: Determination of the structure of integral membrane proteins is a challenging task that is essential to understand how fundamental biological processes (such as photosynthesis, respiration and solute translocation) function at the atomic level. Crystallisation of membrane proteins in 3D has led to the determination of four atomic resolution structures [photosynthetic reaction centres (Allen et al. 1987; Chang et al. 1991; Deisenhofer & Michel, 1989; Ermler et al. 1994); porins (Cowan et al. 1992; Schirmer et al. 1995; Weiss et al. 1991); prostaglandin H2 synthase (Picot et al. 1994); light harvesting complex (McDermott et al. 1995)], and crystals of membrane proteins formed in the plane of the lipid bilayer (2D crystals) have produced two more structures [bacteriorhodopsin (Henderson et al. 1990); light harvesting complex (Kuhlbrandt et al. 1994)].

An investigation of the role of transmembrane domains in Golgi protein retention.

Abstract: The single transmembrane domains (TMDs) of the resident glycosylation enzymes of the Golgi apparatus are involved in preventing these proteins moving beyond the Golgi. It has been proposed that either the TMDs associate, resulting in the formation of large oligomers of Golgi enzymes, or that they mediate the lateral segregation of the enzymes between lipid microdomains. Evidence for either type of interaction has been sought by examining the retention of sialyltransferase (ST), an enzyme of the mammalian trans Golgi. No evidence could be obtained for specific interactions or 'kin recognition' between ST and other proteins of the trans Golgi. Moreover, it is shown that the previously described kin recognition between enzymes of the medial Golgi involves the lumenal portions of these proteins rather than their TMDs. To investigate further the role of the ST TMD, the effects on Golgi retention of various alterations in the TMD were examined. The addition or removal of residues showed that the efficiency of retention of ST is related to TMD length. Moreover, when a type I plasma membrane protein was expressed with a synthetic TMD of 23 leucines it appeared on the cell surface, but when the TMD was shortened to 17 leucines accumulation in the Golgi was observed. These observations are more consistent with lipid-based sorting of ST TMD, but they also allow for reconciliation with the kin recognition model which appears to act on sequences outside of the TMD.

The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster.

Abstract: We describe the cloning, expression and phenotypic characterisation of repo, a gene from Drosophila melanogaster that is essential for the differentiation and maintenance of glia function. It is not, however, required for the initial determination of glial cells. In the embryo, the gene, which encodes a homeodomain protein, is expressed exclusively in all developing glia and closely related cells in both the central and peripheral nervous systems. The only observed exceptions in the CNS are the midline glia derived from the mesectoderm and two of three segmental nerve root glial cells. Using a polyclonal antibody we traced the spatial and temporal pattern of the protein expression in detail. Embryos homozygous for null alleles of the protein exhibit late developmental defects in the nervous system, including a reduction in the number of glial cells, disrupted fasciculation of axons, and the inhibition of ventral nerve cord condensation. The expression of an early glial-specific marker is unaffected in such homozygotes. By contrast, the expression of late glial-specific markers is either substantially reduced or absent. The specificity of expression is also observed in the locust Schistocerca gregaria and is thus evolutionarily conserved.

Distinct spatial and temporal expression patterns of two type I receptors for bone morphogenetic proteins during mouse embryogenesis

Abstract: Bone morphogenetic proteins (BMPs) are multifunctional proteins structurally related to transforming growth factor-beta (TGF beta) and activin that can induce cartilage and bone growth in vivo. Members of the TGF beta superfamily exert their biological effects via heteromeric serine/threonine kinase complexes of type I and type II receptors. We previously obtained six different type I receptors, termed activin receptor-like kinase-1 (ALK-1) to -6. ALK-5 is a TGF beta type I receptor, ALK-2 and ALK-4 are activin type I receptors, and ALK-3 and ALK-6 are type I receptors for osteogenic protein-1 (OP-1)/bone morphogenetic protein-7 (BMP-7) and BMP-4. Here we report the complementary DNA cloning of the mouse homolog of ALK-3, which is highly conserved between mouse and man. ALK-3 messenger RNA (mRNA) is ubiquitously expressed in various adult mouse tissues, whereas ALK-6 mRNA is only found in brain and lung. The distribution of ALK-3 and ALK-6 mRNA in the postimplantation mouse embryo [6.5-15.5 days postcoitu...

Exceptionally Stable Nucleic Acid Hairpins

Abstract: Hairpins represent the dominant secondary structure element in RNA. Certain sequences are found with exceptional frequency in many RNAs and are characterized by exceptionally high thermodynamic stability. Stable RNA hairpins define nucleation sites for folding, determine tertiary interactions in RNA enzymes, protect mRNAs from degradation, and are recognized by RNA-binding proteins. The structures of several stable DNA and RNA hairpins have revealed networks of stabilizing interactions within the hairpin loop: non-Watson-Crick base pairs and base-phosphate and base-sugar contacts. The unusual stability of these structural elements can be used to stabilize RNA and DNA structures and to protect antisense oligonucleotides and mRNAs against exonucleolytic degradation.

A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination.

Abstract: We have developed a strategy for chromosome engineering in embryonic stem (ES) cells that relies on sequential gene targeting and Cre-loxP site-specific recombination. Gene targeting was first used to integrate loxP sites at the desired positions in the genome. Transient expression of Cre recombinase was then used to mediate the chromosomal rearrangement. A genetic selection relying on reconstruction of a selectable marker from sequences co-integrated with the loxP sites allowed detection of cells containing the Cre-mediated rearrangement. A programmed translocation between the c-myc and immunoglobulin heavy chain genes on chromosomes 15 and 12 was created by this method. This strategy will allow the design of a variety of chromosome rearrangements that can be selected and verified in ES cells or activated in ES cell-derived mice.

Antibody VH Domains as Small Recognition Units

Abstract: To develop immunoglobulin based recognition units of minimum size, a human heavy chain variable domain (VH) was designed for selection of phage displayed VH. Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface. These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners. The third hypervariable loop of the modified VH was then randomised to yield a repertoire of 2 x 10(8) independent clones, which was displayed on phage and selected through antigen binding. VH clones specific for hapten and protein antigens were isolated. Soluble VH was expressed with an isoleucine residue at position 47 to improve expression and stability compared to VH containing a glycine residue at this position, which however was preferable for phage selection. Affinities of soluble VH for hapten were between 100 nM and 400 nM. The VH domains were highly specific, stable and well expressed in Escherichia coli. These positive biophysical properties and their small size make them attractive for biotechnological applications.

Inhibition of Drosophila EGF receptor activation by the secreted protein Argos

Abstract: THE Drosophila homologue of the mammalian epidermal growth factor (EGF) receptor (DER)1,2 is a receptor tyrosine kinase involved in many stages of fly development, including photoreceptor determination, and wing-vein formation3–9. Its primary activating ligand is the Spitz protein10,11, which is similar to mammalian TGF-α (ref. 12). Argos is a secreted protein that, like Spitz, contains a single EGF motif13–15. It is a represser of cell determination in the eye, and acts in other tissues, including the wing16–18. Because Argos has the opposite effects to DER in the eye (the former blocks photoreceptor determination, the latter promotes it) we have tested whether it acts by blocking the DER pathway. We show that Argos does indeed repress this pathway in vivo and find that, in vitro, Argos protein can inhibit the activation of DER by Spitz. Thus the determination of cells by the DER pathway is regulated by a balance between extracellular activating and inhibiting signals. This is the first in vivo example of an extracellular inhibitor of a receptor tyrosine kinase.

Targeting of non-Ig sequences in place of the V segment by somatic hypermutation.

Abstract: Affinity maturation of antibodies is characterized by localized hypermutation of the DNA around the V segment. Here we show, using mice containing single or multiple transgene constructs, that an immunoglobulin V kappa segment can be replaced by human beta-globin or prokaryotic neo or gpt genes without affecting the rate of hypermutation; the V gene itself is not necessary for recruiting hypermutation. The ability to target hypermutation to heterologous genes in vivo could find more general applications in biology.

A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli.

Abstract: The regulation of capsular polysaccharide synthesis in Escherichia coli K-12 depends on the level of an unstable positive regulator, RcsA. The amount of RcsA protein is limited both by its rapid degradation by Lon, an ATP-dependent protease, and by its low level of synthesis. We have found that the low level of expression from the rcsA promoter is due to transcriptional silencing by the histone-like protein H-NS; this silencing is sensitive to both sequence and context in a region upstream of the -35 region of the promoter. A small (85-nt) RNA, DsrA, when overproduced, activates transcription of rcsA::lacZ fusions by counteracting H-NS silencing. DsrA RNA does not show any extended homology with the rcsA promoter or other sequenced regions of E. coli. Since the stimulation of rcsA transcription by this small RNA does not depend on any sequences from within the rcsA transcript, DsrA acts, either directly or indirectly, on rcsA transcription initiation.