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PubMed2005

Array-based comparative genomic hybridization analysis of recurrent chromosome 15q rearrangements.

Genomic rearrangements of chromosome 15q11-q13 cause diverse phenotypes including autism, Prader-Willi syndrome (PWS), and Angelman syndrome (AS). This region is subject to genomic imprinting and characterized by complex combinations of low copy repeat elements. Prader-Willi and Angelman syndrome are caused primarily by 15q11-13 deletions of paternal and maternal origin, respectively. Autism is seen with maternal, but not paternal, interstitial duplications. Isodicentric 15q, most often of maternal origin, is associated with a complex phenotype often including autistic features. Limitations of conventional cytogenetic tests preclude a detailed analysis in most patients with 15q rearrangements. We have developed a microarray for comparative genomic hybridization utilizing 106 genomic clones from chromosome 15q to characterize this region. The array accurately localized all breakpoints associated with gains or losses on 15q. The results confirmed the location of the common breakpoints associated with interstitial deletions and duplications. The majority of idic(15q) chromosomes are comprised of symmetrical arms with four copies of the breakpoint 1 to breakpoint 5 region. Patients with less common breakpoints that are not distinguished by routine cytogenetic methods were more accurately characterized by array analysis. This microarray provides a detailed characterization for chromosomal abnormalities involving 15q11-q14 and is useful for more precise genotype-phenotype correlations for autism, PWS, AS, and idic(15) syndrome.

Chromosome Aberrations↗

PubMed1997

Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI.

Exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex has been visualized in living cells using a chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFP[ct]). Upon shifting to permissive temperature, ts-G-GFP(ct) concentrates into COPII-positive structures close to the ER, which then build up to form an intermediate compartment or transport complex, containing ERGIC-53 and the KDEL receptor, where COPII is replaced by COPI. These structures appear heterogenous and move in a microtubule-dependent manner toward the Golgi complex. Our results suggest a sequential mode of COPII and COPI action and indicate that the transport complexes are ER-to-Golgi transport intermediates from which COPI may be involved in recycling material to the ER.

Animals↗

PubMed1994

Domain structures of the MS ring component protein (FliF) of the flagellar basal body of Salmonella typhimurium.

The proximal end of the flagellar basal body consists of a structure called the MS ring complex: two (M and S) rings with different thicknesses closely apposed and a rod extending from the center of the S ring. It has been shown that the MS ring complex consists of multiple copies of single protein FliF (molecular mass 61 kDa). We analyzed the domains of FliF to elucidate how a single protein can be used to construct a complicated particle with several distinct sub-structures. Tryptic digestion of the MS ring complex gave rise to a structure which lacked most of the M ring portion by electron microscopy and showed a major band at 25 kDa by SDS/gel electrophoresis. Amino acid sequence analysis of this band showed that both terminal regions of FliF have been digested, leaving a semi-stable peptide starting from Phe120 and ending at around 400. In addition, we constructed a truncated fliF gene which encodes a FliF lacking 103 amino acid residues from the C terminus. Amplification of the truncated FliF gave rise to a ring complex lacking the rim of the M ring. From these results we assign both terminal regions of FliF to the M ring. Possible domain structures of FliF corresponding to the S ring and the rod are also discussed.

Amino Acid Sequence↗

PubMed1991

IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting.

Multiple copies of an insertion sequence, IS6110, were shown to be present in the genome of members of the Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis). Ten to 12 copies are present in various strains of M. tuberculosis, while strains of M. bovis contain only one to three copies. IS6110 was not detected in the DNA of other species of mycobacteria. Restriction endonuclease analysis indicated that the sequence of IS6110 is conserved across strain and species lines. Hybridization to the insertion sequence can be used to detect restriction fragment length polymorphism reflecting divergence in the sequence of regions flanking the various copies of IS6110. These differences were used to fingerprint various strains of the M. tuberculosis complex.

Autoradiography↗

PubMed2002

Identification of Sec36p, Sec37p, and Sec38p: components of yeast complex that contains Sec34p and Sec35p.

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37 or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4, COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Biochemical characterisation of the clamp/clamp loader proteins from the euryarchaeon Archaeoglobus fulgidus.

Replicative polymerases of eukaryotes, prokaryotes and archaea obtain processivity using ring-shaped DNA sliding clamps that are loaded onto DNA by clamp loaders [replication factor C (RFC) in eukaryotes]. In this study, we cloned the two genes for the subunits of the RFC homologue of the euryarchaeon Archaeoglobus fulgidus. The proteins were expressed and purified from Escherichia coli both individually and as a complex. The afRFC subunits form a heteropentameric complex consisting of one copy of the large subunit and four copies of the small subunits. To analyse the functionality of afRFC, we also expressed the A.fulgidus PCNA homologue and a type B polymerase (PolB1) in E.coli. In primer extension assays, afRFC stimulated the processivity of afPolB1 in afPCNA-dependent reactions. Although the afRFC complex showed significant DNA-dependent ATPase activity, which could be further stimulated by afPCNA, neither of the isolated afRFC subunits showed this activity. However, both the large and small afRFC subunits showed interaction with afPCNA. Furthermore, we demonstrate that ATP binding, but not hydrolysis, is needed to stimulate interactions of the afRFC complex with afPCNA and DNA.

Adenosine Triphosphatases↗

PubMed2000

Molecular characterization of the yeast vacuolar H+-ATPase proton pore.

The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases. The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation. The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides. Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803). Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p. Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain. Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p. Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase.

Escherichia coli↗

PubMed2006

Comparability of the Rey-Osterrieth Complex Figure, the Taylor Complex Figure, and the Modified Taylor Complex Figure in a normal sample of Japanese speakers.

The comparability of recall performance was examined on the Rey-Osterrieth Complex Figure, the Taylor Complex Figure, and the Modified Taylor Complex Figure as measures of visuospatial abilities and visual memory in 66 healthy Japanese university students. A between-subjects design was used in which each of the participants received the three figures. The interrater reliability of each was excellent. While the three figures yielded equivalent copy scores, the Taylor Complex Figure produced significantly higher scores than the other two on both short (3-min.) and delayed (30-min.) recall tests. There were no significant differences between recall scores of the Rey-Osterrieth and the Modified Taylor figures. These results are consistent with the findings of the 2002 study of English-speakers by Hubley and Tremblay, and support that the Modified Taylor Figure is superior to the original one for the companion figure to the Rey-Osterrieth Complex Figure.

Adolescent↗

PubMed1994

Organization of the human genome.

The human genome contains about 3 x 10(9) base pairs which, based on average estimates for the size of coding regions, might suggest an upper limit of about 3 million genes. Evidence from a variety of approaches indicates a much lower figure--in the range 40,000-100,000 genes. Renaturation kinetic analysis reveals the presence of many repetitive elements in the human genome. Some of these are quite simple and highly reiterated, others are more complex and exist in fewer copies. The distribution and function of some of these families and the evolution of complex genomes from simpler ones are discussed.

Chromosome Mapping↗

PubMed2003

Inter- and intra-octarepeat Cu(II) site geometries in the prion protein: implications in Cu(II) binding cooperativity and Cu(II)-mediated assemblies.

Cu(II) binding to the alpha prion protein (alphaPrP) can be both intramolecular and intermolecular. X-ray absorption spectroscopy at the copper K-edge has been used to explore the site geometry under each binding mode using both insoluble polymeric Cu(II).alphaBoPrP-(24-242) (bovine PrP) complexes and soluble Cu(II) complexes of peptides containing one, two, and four copies of the octarepeat. Analysis of the extended region of the spectra using a multiple scattering approach revealed two types of sites differing in the number of His residues in the first coordination shell of Cu(II). Peptides containing one and two-octarepeat copies in sub-stoichiometric Cu(II) complexes showed the direct binding of a single His in accord with crystallographic intra-repeat geometry. Alternatively, the polymeric Cu(II).alphaBoPrP-(24-242) complex and Cu(II) in its soluble complex with a four-octarepeat peptide at half-site-occupancy showed Cu(II) directly bound to two His residues, consistent with an inter-repeat binding mode. Increasing the Cu(II) site occupancy from 0.5 to 0.75 in the peptide containing four octarepeats resulted in spectral features that are intermediate to those of the inter- and intra-repeat modes. The transition from His-Cu-His (inter-repeat) to Cu-His (intra-repeat) on increasing Cu(II) saturation offers a structural basis for the positive cooperativity of the cation binding process and explains the capacity of alphaPrP to participate in Cu(II)-mediated intermolecular interactions.

Amino Acid Sequence↗

PubMed2005

Crystal structure of isoaspartyl aminopeptidase in complex with L-aspartate.

The crystal structure of Escherichia coli isoaspartyl aminopeptidase/asparaginase (EcAIII), an enzyme belonging to the N-terminal nucleophile (Ntn)-hydrolases family, has been determined at 1.9-A resolution for a complex obtained by cocrystallization with l-aspartate, which is a product of both enzymatic reactions catalyzed by EcAIII. The enzyme is a dimer of heterodimers, (alphabeta)(2). The (alphabeta) heterodimer, which arises by autoproteolytic cleavage of the immature protein, exhibits an alphabetabetaalpha-sandwich fold, typical for Ntn-hydrolases. The asymmetric unit contains one copy of the EcAIII.Asp complex, with clearly visible l-aspartate ligands, one bound in each of the two active sites of the enzyme. The l-aspartate ligand is located near Thr(179), the N-terminal residue of subunit beta liberated in the autoproteolytic event. Structural comparisons with the free form of EcAIII reveal that there are no major rearrangements of the active site upon aspartate binding. Although the ligand binding mode is similar to that observed in an l-aspartate complex of the related enzyme human aspartylglucosaminidase, the architecture of the EcAIII active site sheds light on the question of substrate specificity and explains why EcAIII is not able to hydrolyze glycosylated asparagine substrates.

Amino Acid Sequence↗

PubMed2005

Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore.

The Ndc80 complex is a constituent of the outer plate of the kinetochore and plays a critical role in establishing the stable kinetochore-microtubule interactions required for chromosome segregation in mitosis. The Ndc80 complex is evolutionarily conserved and contains the four subunits Spc24, Spc25, Nuf2, and Ndc80 (whose human homologue is called Hec1). All four subunits are predicted to contain globular domains and extensive coiled coil regions. To gain an insight into the organization of the human Ndc80 complex, we reconstituted it using recombinant methods. The hydrodynamic properties of the recombinant Ndc80 complex are identical to those of the endogenous HeLa cell complex and are consistent with a 1:1:1:1 stoichiometry of the four subunits and a very elongated shape. Two tight Hec1-Nuf2 and Spc24-Spc25 subcomplexes, each stabilized by a parallel heterodimeric coiled coil, maintain this organization. These subcomplexes tetramerize via an interaction of the C- and N-terminal portions of the Hec1-Nuf2 and Spc24-Spc25 coiled coils, respectively. The recombinant complex displays normal kinetochore localization upon injection in HeLa cells and is therefore a faithful copy of the endogenous Ndc80 complex.

Cell Cycle Proteins↗

PubMed2005

Cross-cultural differences for three visual memory tasks in Brazilian children.

Norms for three visual memory tasks, including Corsi's block tapping test and the BEM 144 complex figures and visual recognition, were developed for neuropsychological assessment in Brazilian children. The tasks were measured in 127 children ages 7 to 10 years from rural and urban areas of the States of São Paulo and Minas Gerais. Analysis indicated age-related but not sex-related differences. A cross-cultural effect was observed in relation to copying and recall of Complex pictures. Different performances between rural and urban children were noted.

Age Factors↗

PubMed2005

Sotos syndrome common deletion is mediated by directly oriented subunits within inverted Sos-REP low-copy repeats.

Sotos syndrome (Sos) is an overgrowth disorder also characterized clinically by mental retardation, specific craniofacial features and advanced bone age. As NSD1 haploinsufficiency was determined in 2002 to be the major cause of Sos, many intragenic mutations and chromosomal microdeletions involving the entire NSD1 gene have been described. In the Japanese population, half of the cases analyzed appear to have a common microdeletion; however, in the European population, deletion cases account for only 9%. Blast analysis of the Sos genomic region on 5q35 revealed two complex mosaic low-copy repeats (LCRs) that are centromeric and telomeric to NSD1. We termed these proximal Sos-REP (Sos-PREP, approximately 390 kb) and distal Sos-REP (Sos-DREP, approximately 429 kb), respectively. On the basis of the analysis of DNA sequence, we determined the size, structure, orientation and extent of sequence identity of these LCRs. We found that Sos-PREP and Sos-DREP are composed of six subunits termed A-F. Each of the homologous subunits, with the exception of one, is located in an inverted orientation and the order of subunits is different between the two Sos-REPs. Only the subunit C' in Sos-DREP is oriented directly with respect to the subunit C in Sos-PREP. These latter C' and C subunits are greater than 99% identical. Using pulsed-field gel electrophoresis analysis in eight Sos patients with a common deletion, we detected an approximately 550 kb junction fragment that we predicted according to the non-allelic homologous recombination (NAHR) mechanism using directly oriented Sos-PREP C and Sos-DREP C' subunits as substrates. This patient specific junction fragment was not present in 51 Japanese and non-Japanese controls. Subsequently, using long-range PCR with restriction enzyme digestion and DNA sequencing, we identified a 2.5 kb unequal crossover hotspot region in six out of nine analyzed Sos patients with the common deletion. Our data are consistent with an NAHR mechanism for generation of the Sos common deletion.

Centromere↗

PubMed2000

E4 allele dosage does not predict cholinergic activity or synapse loss in Alzheimer's disease.

OBJECTIVE: To investigate the relationship between apolipoprotein E (APOE) genotype and both cholinergic dysfunction and synapse loss in AD. BACKGROUND: A reduction in neocortical synapses and marked losses in the cholinergic system occur in AD. It has been suggested that the number of APOE epsilon4 alleles is inversely related to choline acetyltransferase (ChAT) activity, thereby influencing cholinergic function. Whether APOE genotype may influence neocortical synapse loss remains unclear. METHODS: An autopsy series of 182 patients with AD (National Institute on Aging and Consortium to Establish a Registry for Alzheimer's Disease criteria) and 16 normal controls (NC). APOE genotype was determined in blood samples or in postmortem brain tissue. Midfrontal synapse counts (AU/microg) were quantified by a dot-immunobinding assay for synaptophysin (Syn). Midfrontal ChAT activity (nmol/h/100 mg) was assessed using standard assays. RESULTS: Mean midfrontal ChAT activity and Syn were both significantly reduced in patients with AD compared with NC. The relationship between ChAT activity and number of epsilon4 allele copies in AD was complex, with ChAT activity lower in patients with either two or no epsilon4 alleles compared with those with one epsilon4 allele. There was no relationship between APOE genotype and synapse loss in AD. Syn density was almost identical across the three genotypes. CONCLUSIONS: Unlike other studies, we failed to detect a linear relationship between ChAT activity and number of epsilon4 allele copies in the midfrontal cortex of this large sample of patients with AD. Our data also show that the presence of epsilon4 allele does not influence midfrontal synapse loss in AD. This suggests that factors other than APOE genotype may be operative in the decline in midfrontal cholinergic function and synapses seen in AD.

Aged↗

PubMed1978

Sequence complexity of nuclear and polysomal RNA in leaves of the tobacco plant.

The first measurements are reported of the sequence complexity of nuclear and polysomal RNA contained within the cells of a higher plant. Polysomal RNA from tobacco leaves was prepared by a procedure which minimized contamination with nuclear RNA. Hybridization of 3H-cDNA complementary to polysomal poly(A) RNA with an excess of tobacco DNA indicated that greater than 95% of the poly(A) mRNA was transcribed from single-copy sequences. RNA excess hybridization reactions with polysomal poly(A) RNA and 3H-cDNA revealed the presence of three abundance classes in the poly(A) mRNA. The best least-squares solution indicated that these classes comprise 9, 52, and 39% of the poly (A) mRNA and contain sequences present an average of 4500, 340 and 17 times per cell. Hybridization reactions containing an excess of nuclear or total polysomal RNA and 3H-single-copy DNA indicated the complexity of these RNA populations to be 1.19 X 10(8) nucleotides (nuclear) and 3.33 X 10(7) nucleotides (polysomal). Thus only 28% of the nuclear RNA sequence diversity (27,000 average-sized mRNA sequences) is respresented in leaf polysomes. These results suggest that there is a general similarity in the basic transcriptional processes of metaphytan and metazoan cells.

Base Sequence↗

PubMed2006

Small regions of overlapping deletions on 6q26 in human astrocytic tumours identified using chromosome 6 tile path array-CGH.

Deletions of chromosome 6 are a common abnormality in diverse human malignancies including astrocytic tumours, suggesting the presence of tumour suppressor genes (TSG). In order to help identify candidate TSGs, we have constructed a chromosome 6 tile path microarray. The array contains 1,780 clones (778 P1-derived artificial chromosome and 1,002 bacterial artificial chromosome) that cover 98.3% of the published chromosome 6 sequences. A total of 104 adult astrocytic tumours (10 diffuse astrocytomas, 30 anaplastic astrocytomas (AA), 64 glioblastomas (GB)) were analysed using this array. Single copy number change was successfully detected and the result was in general concordant with a microsatellite analysis. The pattern of copy number change was complex with multiple interstitial deletions/gains. However, a predominance of telomeric 6q deletions was seen. Two small common and overlapping regions of deletion at 6q26 were identified. One was 1,002 kb in size and contained PACRG and QKI, while the second was 199 kb and harbours a single gene, ARID1B. The data show that the chromosome 6 tile path array is useful in mapping copy number changes with high resolution and accuracy. We confirmed the high frequency of chromosome 6 deletions in AA and GB, and identified two novel commonly deleted regions that may harbour TSGs.

Astrocytoma↗

PubMed1999

Clathrin and two components of the COPII complex, Sec23p and Sec24p, could be involved in endocytosis of the Saccharomyces cerevisiae maltose transporter.

The Saccharomyces cerevisiae maltose transporter is a 12-transmembrane segment protein that under certain physiological conditions is degraded in the vacuole after internalization by endocytosis. Previous studies showed that endocytosis of this protein is dependent on the actin network, is independent of microtubules, and requires the binding of ubiquitin. In this work, we attempted to determine which coat proteins are involved in this endocytosis. Using mutants defective in the heavy chain of clathrin and in several subunits of the COPI and the COPII complexes, we found that clathrin, as well as two cytosolic subunits of COPII, Sec23p and Sec24p, could be involved in internalization of the yeast maltose transporter. The results also indicate that endocytosis of the maltose transporter and of the alpha-factor receptor could have different requirements.

Biological Transport↗