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PubMed2006

Evolution of intraflagellar transport from coated vesicles and autogenous origin of the eukaryotic cilium.

The cilium/flagellum is a sensory-motile organelle ancestrally present in eukaryotic cells. For assembly cilia universally rely on intraflagellar transport (IFT), a specialised bidirectional transport process mediated by the ancestral and conserved IFT complex. Based on the homology of IFT complex proteins to components of coat protein I (COPI) and clathrin-coated vesicles, we propose that the non- vesicular, membrane-bound IFT evolved as a specialised form of coated vesicle transport from a protocoatomer complex. IFT thus shares common ancestry with all protocoatomer derivatives, including all vesicle coats and the nuclear pore complex (NPC). This has major implications for the evolutionary origin of the cilium. First, it reinforces the tenet that duplication and divergence of pre-existing structures, rather than symbiosis, were the major themes during cilium evolution. Second, it suggests that the initial step in the autogenous origin of the cilium was the establishment of a membrane patch with transmembrane proteins transported by the ancestral vesicle-coating IFT complex. We propose a scenario for how the initial membrane patch gradually protruded to enhance exposure to the environment, then started to move, and finally compartmentalised to render receptor signalling and ciliary beating more efficient.

Animals↗

PubMed1993

Formaldehyde lacks genotoxicity in formaldehyde-hyperresistant strains of the yeast Saccharomyces cerevisiae.

Formaldehyde is a weak mutagen and recombinagen in wild type strains of Saccharomyces cerevisiae. Genotoxicity of formaldehyde is influenced by the activity of the SFA yeast gene. Yeast transformants containing multiple copies of the plasmid-contained SFA gene are hyperresistant to the chemical and grow in the presence of 5 mM formaldehyde, a concentration lethal for the wild type. The SFA-encoded protein mediates the degradation of formaldehyde and its activity is most probably responsible for the low or absent genotoxic effects in formaldehyde-treated cells. Multi-copy vectors containing the SFA gene are retained in yeast transformants growing in complex media supplemented with 5 mM formaldehyde. Cells harboring such multi-copy vectors may, therefore, be propagated in large batch cultures in undefined media in the presence of formaldehyde concentrations toxic to the wild type.

Drug Resistance↗

PubMed1997

Tricorn protease exists as an icosahedral supermolecule in vivo.

Tricorn protease is the core enzyme of a recently discovered modular proteolytic system. We present evidence that tricorn protease exists in vivo in the form of a higher-order assembly, namely as an icosahedral capsid. Its size exceeds that of many virus particles and represents by far the largest known homooligomeric enzyme complex. Each capsid is built from 20 copies of the tricorn hexameric toroid and thus has a molecular weight of 14.6 MDa. Three-dimensional reconstructions of ice-embedded capsids from electron micrographs show that it is hollow and has large void volumes in its wall. We suggest that the tricorn capsid, in addition to its intrinsic proteolytic activity, serves as the organizing center of a multienzyme complex.

Archaeal Proteins↗

PubMed2000

Genetic imbalances in precursor lesions of endometrial cancer detected by comparative genomic hybridization.

Endometrial hyperplasia is regarded as a precursor lesion of endometrioid adenocarcinomas of the endometrium. The genetic events involved in the multistep process from normal endometrial glandular tissue to invasive endometrial carcinomas are primarily unknown. We chose endometrial hyperplasia as a model for identifying chromosomal aberrations occurring during carcinogenesis. Comparative genomic hybridization (CGH) was performed on 47 formalin-fixed, paraffin-embedded specimens of endometrial hyperplasia using the microdissection technique to increase the number of tumor cells in the samples and reduce contamination from normal cells. CGH analysis revealed that 24 out of 47 (51%) samples had detectable chromosomal imbalances, whereas 23 (49%) were in a genetically balanced state. The incidence of aberrant CGH profiles tended to parallel dysplasia grade, ranging from 22% aberrant profiles in simple hyperplasia to 67% in complex hyperplasia with atypia. The most frequent imbalances were 1p, 16p, and 20q underrepresentations and 4q overrepresentations. Copy number changes in 1p were more frequent in atypical complex hyperplasia than in complex lesions without atypical cells or simple lesions (42% versus 20% and 0%). Our results show that endometrial hyperplasia reveals recurrent chromosomal imbalances which tend to increase with the presence of atypical cells. The most frequent aberrations in endometrial cancer, 1q and 8q overrepresentations, are not present or are rare in its precursor lesions. This analysis provides evidence that tumorigenesis proceeds through the accumulation of a series of genetic alterations and suggests a stepwise mode of tumorigenesis.

Adenocarcinoma↗

PubMed2005

Gene expression analysis of the function of the male-specific lethal complex in Drosophila.

Dosage compensation refers to the equal expression of X-linked genes despite the difference in copy number between the two sexes. The male-specific lethal (MSL) complex is concentrated on the X chromosome in males. A gene expression assay for embryos was developed to examine the function of this complex. In mutant male embryos without either the MSL complex or MOF histone acetylase, dosage compensation is retained but autosomal expression is increased. Dosage compensation is lost in the double-mutant embryos. In embryos in which the MSL complex and MOF are targeted to the X chromosomes in females, the results are consistent with previous surveys showing that in general the X expression remains unchanged, but autosomal expression is reduced. Mutations in the ISWI chromatin-remodeling component cause increases specifically of X-linked genes in males. Thus, the function of the MSL complex in conjunction with ISWI is postulated to override the effect on gene expression of high histone acetylation on the male X. The basic determinant of dosage compensation is suggested to be an inverse dosage effect produced by an imbalance of transcription factors on the X vs. the autosomes. The sequestration of the MSL complex to the male X may have evolved to counteract a similar effect on the autosomes and to prevent an overexpression of the X chromosome in males that would otherwise occur due to the high levels of histone acetylation.

Acetyltransferases↗

PubMed2006

The Plasmodium falciparum RhopH2 promoter and first 24 amino acids are sufficient to target proteins to the rhoptries.

The rhoptry secretory organelles of the malaria parasite, Plasmodium falciparum, contain a RhopH complex, which is composed of the proteins RhopH1, RhopH2, and RhopH3. RhopH1 is encoded by the rhoph1/clag multi-gene family, whereas RhopH2 and RhopH3 are encoded by single-copy genes. The precise function of the RhopH complex has not been identified, but it has been shown that the component proteins are involved in erythrocyte binding and perhaps participate in the formation of the parasitophorous vacuolar membrane. In this study, we have isolated pfrhoph2 promoter plus the signal peptide encoding sequence and generated transgene expression constructs to evaluate a trafficking and the RhopH complex formation in transgenic P. falciparum parasite lines. Interestingly, we found that the N-terminal 24 amino acids of RhopH2, including signal peptide sequence, were sufficient to target GFP to the rhoptries under the rhoph2 promoter. Because it was previously shown that the timing of the expression alone could not target proteins to the apical organelles, this targeting is likely mediated via a unique mechanism that is dependent on N-terminal 24 amino acids of RhopH2 early in the secretory pathway. The N-terminal one third of Clag3.1, which contains a distinct conserved domain with Toxoplasma gondii RON2, can not associate the RhopH complex as a GFP chimera, but a c-Myc-Clag3.1 chimera lacking the C-terminus successfully associates the RhopH complex indicating that cooperation of middle region is likely required but the C-terminus is not necessary.

Amino Acid Sequence↗

PubMed1998

Adjustable forming of thermoplastic composites for orthopaedic applications.

The present study was focused on the development of a special thermoforming technique for manufacturing of continuous fibre reinforced thermoplastic composite parts with complex surface contours. In particular, a stamp forming process was modified to investigate the potential manufacturing advantages of thermoplastic composites in orthopaedic applications. An apparatus was designed which allowed the thermoforming procedure to be fully automatic, i.e. a cold pre-consolidated laminate panel, as the feed material, was heated up in an infrared heating zone and then transferred into a cold mould system, where it was stamp formed. Both halves of the mould were made of many tiny round metal sticks in a metal frame. This needle-bed mould allowed one to copy any contour by pushing it slightly on spring fixed sticks. The desired position of these sticks could then be adjusted by forcing the side plates of the metal frame together. To prevent any press mark of stick-tops on the composite, i.e. to achieve smooth surfaces of the themoformed composite parts, flexible rubber pads were needed to cover the mould surfaces. Experimental results showed that the surface profile of CF/PP and GF/PP composites formed by the needle-bed mould reproduced fairly well the contour of a saddle shaped, complex model sample. Unique properties of this needle-bed mould are that it can be repeatedly used, and that it can copy any complex surface contours, for example a bone surface, by simply adjusting the stick positions according to the special surface requirements.

Journal Article↗

PubMed1999

Role of microtubules in the organization of the Golgi complex.

The Golgi complex of mammalian cells is composed of cisternal stacks that function in processing and sorting of membrane and luminal proteins during transport from the site of synthesis in the endoplasmic reticulum to lysosomes, secretory vacuoles, and the cell surface. Even though exceptions are found, the Golgi stacks are usually arranged as an interconnected network in the region around the centrosome, the major organizing center for cytoplasmic microtubules. A close relation thus exists between Golgi elements and microtubules (especially the stable subpopulation enriched in detyrosinated and acetylated tubulin). After drug-induced disruption of microtubules, the Golgi stacks are disconnected from each other, partly broken up, dispersed in the cytoplasm, and redistributed to endoplasmic reticulum exit sites. Despite this, intracellular protein traffic is only moderately disturbed. Following removal of the drugs, scattered Golgi elements move along reassembling microtubules back to the centrosomal region and reunite into a continuous system. The microtubule-dependent motor proteins cytoplasmic dynein and kinesin bind to Golgi membranes and have been implicated in vesicular transport to and from the Golgi complex. Microinjection of dynein heavy chain antibodies causes dispersal of the Golgi complex, and the Golgi complex of cells lacking cytoplasmic dynein is likewise spread throughout the cytoplasm. In a similar manner, kinesin antibodies have been found to inhibit Golgi-to-endoplasmic reticulum transport in brefeldin A-treated cells and scattering of Golgi elements along remaining microtubules in cells exposed to a low concentration of nocodazole. The molecular mechanisms in the interaction between microtubules and membranes are, however, incompletely understood. During mitosis, the Golgi complex is extensively reorganized in order to ensure an equal partitioning of this single-copy organelle between the daughter cells. Mitosis-promoting factor, a complex of cdc2 kinase and cyclin B, is a key regulator of this and other events in the induction of cell division. Cytoplasmic microtubules depolymerize in prophase and as a result thereof, the Golgi stacks become smaller, disengage from each other, and take up a perinuclear distribution. The mitotic spindle is thereafter put together, aligns the chromosomes in the metaphase plate, and eventually pulls the sister chromatids apart in anaphase. In parallel, the Golgi stacks are broken down into clusters of vesicles and tubules and movement of protein along the exocytic and endocytic pathways is inhibited. Using a cell-free system, it has been established that the fragmentation of the Golgi stacks is due to a continued budding of transport vesicles and a concomitant inhibition of the fusion of the vesicles with their target membranes. In telophase and after cytokinesis, a Golgi complex made up of interconnected cisternal stacks is recreated in each daughter cell and intracellular protein traffic is resumed. This restoration of a normal interphase morphology and function is dependent on reassembly of a radiating array of cytoplasmic microtubules along which vesicles can be carried and on reactivation of the machinery for membrane fusion.

Animals↗

PubMed1999

Intermediate length Rieske iron-sulfur protein is present and functionally active in the cytochrome bc1 complex of Saccharomyces cerevisiae.

To investigate the relationship between post-translational processing of the Rieske iron-sulfur protein of Saccharomyces cerevisiae and its assembly into the mitochondrial cytochrome bc1 complex we used iron-sulfur proteins in which the presequences had been changed by site-directed mutagenesis of the cloned iron-sulfur protein gene, so that the recognition sites for the matrix processing peptidase or the mitochondrial intermediate peptidase (MIP) had been destroyed. When yeast strain JPJ1, in which the gene for the iron-sulfur protein is deleted, was transformed with these constructs on a single copy expression vector, mitochondrial membranes and bc1 complexes isolated from these strains accumulated intermediate length iron-sulfur proteins in vivo. The cytochrome bc1 complex activities of these membranes and bc1 complexes indicate that intermediate iron-sulfur protein (i-ISP) has full activity when compared with that of mature sized iron-sulfur protein (m-ISP). Therefore the iron-sulfur cluster must have been inserted before processing of i-ISP to m-ISP by MIP. When iron-sulfur protein is imported into mitochondria in vitro, i-ISP interacts with components of the bc1 complex before it is processed to m-ISP. These results establish that the iron-sulfur cluster is inserted into the apoprotein before MIP cleaves off the second part of the presequence and that this second processing step takes place after i-ISP has been assembled into the bc1 complex.

Amino Acid Sequence↗

PubMed1992

Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.

Recently, the insertion element IS1081 from Mycobacterium bovis was identified. In this study, the usefulness of IS1081 in the epidemiology of tuberculosis was investigated. The host range of this insertion sequence was found to be restricted exclusively to the group of Mycobacterium tuberculosis complex bacteria, whereas none of the 10 mycobacterial species which do not belong to the M. tuberculosis complex contained IS1081-homologous DNA. All 99 M. tuberculosis complex strains investigated carried five or six copies of IS1081, and very limited IS1081-associated restriction fragment length polymorphisms were observed among the strains. Seven different IS1081-containing bands were distinguished in each strain, and the patterns differed only in one or two insertion sequence-containing bands. The banding pattern of M. bovis BCG differed in the presence of a 8.0-kb IS1081-containing PvuII fragment which was absent from all other M. tuberculosis complex strains.

Base Sequence↗

PubMed1998

Increased C-MYC oncogene copy number detected with combined modified comparative genomic hybridization and FISH analysis in a Richter syndrome case with complex karyotype.

Modified comparative genomic hybridization (mCGH) was performed in a Richter syndrome case with a complex karyotype to identify and map gains of DNA sequences with possible importance in the pathogenesis and progression of the tumor. The mCGH analysis revealed a more intense signal on part of the long arm of one pair of chromosomes belonging to group C. The G-banding study showed that the increased DNA-sequence copy number originated from the 8q22-->qter chromosomal region. This increase was confirmed by performing a fluorescence in situ hybridization analysis on tumor metaphases by first using a chromosome 8-specific library and subsequently a C-MYC probe, which revealed positive staining on six different regions located on six different chromosomes, each one bearing a single copy of the C-MYC oncogene. These results show the existence of C-MYC oncogene copy-number increases and confirm the usefulness of mCGH in the genetic analysis of malignancies.

Aged↗

PubMed2004

Novel isotypic gamma/zeta subunits reveal three coatomer complexes in mammals.

In early secretory transport, coat recruitment for the formation of coat protein I (COPI) vesicles involves binding to donor Golgi membranes of the small GTPase ADP-ribosylation factor 1 and subsequent attachment of the cytoplasmic heptameric complex coatomer. Various hypotheses exist as to the precise role of and possible routes taken by COPI vesicles in the mammalian cell. Here we report the ubiquitous expression of two novel isotypes of coatomer subunits gamma- and zeta-COP that are incorporated into coatomer, and show that three isotypes exist of the complex defined by the subunit combinations gamma 1/zeta 1, gamma 1/zeta 2, and gamma 2/zeta 1. In a liver cytosol, these forms make up the total coatomer in a ratio of about 2:1:2, respectively. The coatomer isotypes are located differentially within the early secretory pathway, and the gamma 2/zeta 1 isotype is preferentially incorporated into COPI vesicles. A population of COPI vesicles was characterized that almost exclusively contains gamma 2/zeta 1 coatomer. This existence of three structurally different forms of coatomer will need to be considered in future models of COPI-mediated transport.

Animals↗

PubMed2000

Strain identification of Trichophyton rubrum by specific amplification of subrepeat elements in the ribosomal DNA nontranscribed spacer.

Trichophyton rubrum is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the T. rubrum ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species, Trichophyton violaceum, Trichophyton gourvilii, and Trichophyton soudanense, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing T. rubrum will enable important questions about pathogenesis and epidemiology of this fungus to be addressed.

Base Sequence↗

PubMed1999

Packaging and replication regulation revealed by chimeric genome segments of double-stranded RNA bacteriophage phi6.

Bacteriophage phi6 has a double-stranded RNA genome composed of three linear segments, L, M, and S. The innermost particle in the virion of phi6, like in the other dsRNA viruses, is an RNA-dependent RNA polymerase complex, which carries out all the functions needed for the replication of the viral genome. Empty polymerase complexes can package the single-stranded copies of the viral genome segments, replicate the packaged segments into double-stranded form (minus strand synthesis), and then produce new plus strands (transcripts) from the double-stranded RNA templates. The three viral genomic segments contain unique packaging signals at their 5' ends, and minus strand synthesis initiation is dependent on the sequence at the 3' end. Here we have constructed chimeric segments that have the packaging signal from one segment and the minus strand synthesis initiation signal from another segment. Using purified recombinant polymerase complexes and single-stranded/chimeric and original RNA segments, we have analyzed the packaging and replication regulation operating in in vitro conditions. We show that the 5' end of the L genome segment in single-stranded form is needed to switch from the packaging to the minus strand synthesis and the same sequence is required in double-stranded form to switch on plus strand synthesis. In addition we have constructed deletions to the M segment to analyze the possible regulatory role of the internal noncoding area of this segment.

Bacteriophage phi 6↗

PubMed1990

Characterization of nuclear DNA in 12 species of Chlorella (Chlorococcales, Chlorophyta) by DNA reassociation.

Strains of 12 different species of the genus Chlorella were analyzed for amount, reiteration frequency and kinetic complexity of chromosomal DNA components by C0t analysis. The resulting C0t curves reveal at least two different DNA components consisting of single copy DNA (up to 95%) and of repetitive DNA with complexities of 4.1 x 10(3) base pairs (bp) to approximately 11.7 x 10(3) bp and a reiteration frequency of 100-760. The total amount of repetitive DNA is less than 9% of the nuclear genome and similar in all strains studied. In contrast, the total kinetic complexity varies in a wide range from 1.26 x 10(7) bp to 8.08 x 10(7) bp which is mainly due to differences in the size of single copy DNA. The genome sizes in Chlorella seem not to be correlated with biochemical and physiological characteristics and therefore are unlikely to be useful as a taxonomical marker. A comparison of thermal denaturation profiles showed that the melting points of repetitive and single copy DNA differ by approximately 7 degrees C which may result from base mismatch and/or from a distinct base composition of the repetitive DNA.

Cell Nucleus↗

PubMed1997

Use of organic cosmotropic solutes to crystallize flexible proteins: application to T7 RNA polymerase and its complex with the inhibitor T7 lysozyme.

We have crystallized, using several approaches that may be of general interest, T7 RNA polymerase (T7RP) and the T7 RNA polymerase-T7 lysozyme complex (T7RPL) in forms suitable for structure determination by X-ray crystallography. A series of polyhydric alcohols, sugars, amino and methylamino acids, compounds known to stabilize protein structure, were found to be critical for both crystallization and subsequent improvement of the crystal's diffraction resolution. Moreover, optimal crystallogenesis was achieved through an unconventional "reverse" vapor diffusion sitting drop method that is suitable for proteins that are insoluble at low ionic strength.T7RP has been crystallized in an orthorhombic form (I), space group P222, with cell parameters a=220 A, b=205 A, c=67 A and a monoclinic form (II), space group P21, with cell parameters a=229 A, b=205 A, c=70 A, beta=106 degrees. Crystal form I diffracts X-rays to 3.5 A and form II to 6.0 A. Three and six copies of the polymerase are predicted to be in the asymmetric unit forms I and II, respectively. Three monoclinic crystal forms of the T7RPL complex have been obtained in space group C2. Form I has cell parameters a=320 A, b=93 A, c=229 A, beta=129 degrees, form II has parameters a=293 A, b=93 A, c=68 A, beta=93 degrees, and form III has parameters a=270 A, b=93 A, c=63 A, beta=103 degrees. Crystal form I diffracts synchrotron wiggler radiation to 3.2 A and form III to 2.8 A. Calculations of crystal density imply three or four copies of the complex in form I and one copy in the asymmetric unit of forms II and III.

Amino Acids↗

PubMedDate not supplied

A dumbbell-like complex formation for DNA target assay.

A simple and highly sensitive test for the detection of nucleic acid targets is described. It is based upon complex formation between a small-diameter magnetic particle and a larger and nonmagnetic particle through a hybridization reaction, what we have called a dumbbell-like complex. During the different steps, nonreacting nonmagnetic conjugates were eliminated by magnetic separation. At the end of the process, dumbbell complex number was estimated by counting under a microscope. Compared to the already described two-particle tests, our model was able to reach higher sensitivities, with a threshold typically in the amol/mL range (10(6) copies of HIV DNA/mL) without the need for complex instrumentation or genomic amplification reactions.

Biotin↗

PubMed2006

Hotspots for copy number variation in chimpanzees and humans.

Copy number variation is surprisingly common among humans and can be involved in phenotypic diversity and variable susceptibility to complex diseases, but little is known of the extent of copy number variation in nonhuman primates. We have used two array-based comparative genomic hybridization platforms to identify a total of 355 copy number variants (CNVs) in the genomes of 20 wild-born chimpanzees (Pan troglodytes) and have compared the identified chimpanzee CNVs to known human CNVs from previous studies. Many CNVs were observed in the corresponding regions in both chimpanzees and humans; especially those CNVs of higher frequency. Strikingly, these loci are enriched 20-fold for ancestral segmental duplications, which may facilitate CNV formation through nonallelic homologous recombination mechanisms. Therefore, some of these regions may be unstable "hotspots" for the genesis of copy number variation, with recurrent duplications and deletions occurring across and within species.

Animals↗