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PubMed2002

Molecular analysis of the cytosolic Dictyostelium gamma-tubulin complex.

gamma-Tubulin plays an essential role in microtubule nucleation and organization and occurs, besides its centrosomal localization, in the cytosol, where it forms soluble complexes with other proteins. We investigated the size and composition of gamma-tubulin complexes in Dictyostelium, using a mutant cell line in which the endogenous copy of the gamma-tubulin gene had been replaced by a tagged version. Dictyostelium gamma-tubulin complexes were generally much smaller than the large gamma-tubulin ring complexes found in higher organisms. The stability of the small Dictyostelium gamma-tubulin complexes depended strongly on the purification conditions, with a striking stabilization of the complexes under high salt conditions. Furthermore, we cloned the Dictyostelium homolog of Spc97 and an almost complete sequence of the Dictyostelium homolog of Spc98, which are both components of gamma-tubulin complexes in other organisms. Both proteins localize to the centrosome in Dictyostelium throughout the cell cycle and are also present in a cytosolic pool. We could show that the prevailing small complex present in Dictyostelium consists of DdSpc98 and gamma-tubulin, whereas DdSpc97 does not associate. Dictyostelium is thus the first organism investigated so far where the three proteins do not interact stably in the cytosol.

Amino Acid Sequence↗

PubMed2003

Evidence that the transport of ricin to the cytoplasm is independent of both Rab6A and COPI.

Cholera toxin, Shiga toxin and ricin are examples of protein toxins that require retrograde transport from the Golgi complex into the endoplasmic reticulum (ER) to express their cytotoxic activities and different toxins appear to use different pathways of retrograde transport. Cholera toxin contains the mammalian retrograde targeting signal KDEL and is believed to exploit the coat protein I (COPI) and KDEL receptor-dependent pathway to go from the Golgi complex to the ER. Shiga toxin, however, has no KDEL sequence to specify its inclusion in COPI-coated retrograde vesicles and is believed to use a recently discovered COPI-independent and Rab6A-dependent retrograde pathway to enter the ER. Ricin, like Shiga toxin, does not contain a KDEL sequence and is therefore a candidate to use the COPI-independent and Rab6A-dependent pathway of retrograde transport to access the ER. We measured the effect of the GDP-restricted mutant of Rab6A (Rab6A-T27N) on the cytotoxic activity of ricin and found that expressing Rab6A-T27N in cells did not inhibit the cytotoxicity of ricin, suggesting that ricin enters the cytoplasm by a retrograde pathway that does not involve Rab6A. Moreover, ricin still intoxicated cells when Rab6A and COPI were simultaneously inhibited, implying that ricin requires neither Rab6A nor COPI to intoxicate cells.

Animals↗

PubMed1992

TFC3: gene encoding the B-block binding subunit of the yeast transcription factor IIIC.

Yeast transcription factor IIIC (TFIIIC) is a multisubunit protein complex that interacts with two control elements of class III promoters called the A and B blocks. Here we describe the gene encoding the 138-kDa subunit (tau 138), which is involved in B-block binding. From the DNA sequence, the open reading frame, interrupted by an intron with an unusual 3' splice acceptor site, is in agreement with all the microsequencing data for peptides within tau 138. TFC3 is a single-copy gene located on chromosome I; it is essential for cell viability as shown by a gene disruption experiment. Epitope-tagging of the TFC3 gene product and DNA binding experiments are consistent with the presence of one copy of tau 138 in TFIIIC-DNA complexes.

Amino Acid Sequence↗

PubMed1979

The Friend virus genome: partial characterization of a complete DNA copy.

A complementary DNA probe has been prepared from the Friend murine erythroleukaemia virus complex released by Friend cells (FV cDNAD-) and Friend cells induced to differentiate (FV cDNAD+). Molecular hybridization analysis shows that: (a) FV cDNAD+ is close to being a complete copy of the virus genome and the distribution of sequences is uniform with respect to their distribution in the Friend virus genome. (b) Hybridization of 70S RNA from the cloned helper virus to the total FVc DNAD+ probe demonstrates that a large proportion of the cDNA is specific to the transforming spleen focus forming virus. (c) Hybridization of the probe to normal and transformed cell DNA shows that there are about seven Friend virus related genes in normal DNA and almost twice this amount in transformed cell DNA. A significant minor proportion (20%) of the cDNA probe anneals only to virus related sequences in the transformed cell DNA. (d) An analysis of the kinetics of annealing of the cDNA to an excess template RNA shows that the minimum base sequence complexity of the Friend virus complex is 4 x 10(6). (e) An analysis of the cross hybridization between FV cDNAD+ and 60 to 70S RNA isolated from virus released by uninduced and induced cells shows that the genome of the induced and uninduced Friend virus is almost identical.

Animals↗

PubMed1996

PCR mutagenesis and overexpression of tryptophan synthase from Salmonella typhimurium: on the roles of beta2 subunit Lys-382.

We have devised convenient methods for mutagenesis and very high level expression of wild type and mutant tryptophan synthase alpha and beta2 subunits and alpha2beta2 complex from Salmonella typhimurium. The trpBA genes were modified by introduction of five new restriction sites by polymerase chain reaction (PCR) and were then cloned into the plasmid pTrc99A under trc promoter control. The recombinant plasmid pEBA-10 and three plasmids constructed from pEBA-10 were transformed into Escherichia coli CB149, which lacks tryptophan operon genes. Optimization of growth conditions of the transformed cells resulted in 10- to 40-fold higher yields of cells ( approximately 22 g/liter) than attained previously. The improved expression system gave higher yields of tryptophan synthase proteins (23-70% of the soluble protein) and led to correspondingly high yields of purified alpha and beta2 subunits or alpha2beta2 complex (200-800 mg/liter). A plasmid containing 8 copies of the trpA gene gave the highest yield of alpha subunit. The PCR-based mutagenesis method permits mutation of any base pair in the trpBA genes, between suitable pairs of restriction sites, and requires only one new primer per mutation. The method is illustrated by construction of mutant beta2 subunits with any of five amino acid substitutions at Lys-382, the site of a previously described missense mutation. Characterization of the purified mutant alpha2beta2 complexes shows that Lys-382 in the wild type alpha2beta2 complex does not serve an essential catalytic role but may stabilize an active "closed" conformation of the enzyme by forming a salt bridge with Glu-350.

Bacterial Proteins↗

PubMed2005

Characterization of hCINAP, a novel coilin-interacting protein encoded by a transcript from the transcription factor TAFIID32 locus.

Coilin is a marker protein for the Cajal body, a subnuclear domain acting as a site for assembly and maturation of nuclear RNA-protein complexes. Using a yeast two-hybrid screen to identify coilin-interacting proteins, we have identified hCINAP (human coilin interacting nuclear ATPase protein), a nuclear factor of 172 amino acids with a P-loop nucleotide binding motif and ATPase activity. The hCINAP protein sequence is highly conserved across its full-length from human to plants and yeast and is ubiquitously expressed in all human tissues and cell lines tested. The yeast orthologue of CINAP is a single copy, essential gene. Tagged hCINAP is present in complexes containing coilin in mammalian cells and recombinant, Escherichia coli expressed hCINAP binds directly to coilin in vitro. The 214 carboxyl-terminal residues of coilin appear essential for the interaction with hCINAP. Both immunofluorescence and fluorescent protein tagging show that hCINAP is specifically nuclear and distributed in a widespread, diffuse nucleoplasmic pattern, excluding nucleoli, with some concentration also in Cajal bodies. Overexpression of hCINAP in HeLa cells results in a decrease in the average number of Cajal bodies per nucleus, consistent with it affecting either the stability of Cajal bodies and/or their rate of assembly. The hCINAP mRNA is an alternatively spliced transcript from the TAF9 locus, which encodes the basal transcription factor subunit TAFIID32. However, hCINAP and TAFIID32 mRNAs are translated from different ATG codons and use distinct reading frames, resulting in them having no identity in their respective protein sequences.

Adenosine Triphosphate↗

PubMed2003

Condensin and cohesin: more than chromosome compactor and glue.

Two related protein complexes, cohesin and condensin, are essential for separating identical copies of the genome into daughter cells during cell division. Cohesin glues replicated sister chromatids together until they split at anaphase, whereas condensin reorganizes chromosomes into their highly compact mitotic structure. Unexpectedly, mutations in the subunits of these complexes have been uncovered in genetic screens that target completely different processes. Exciting new evidence is emerging that cohesin and condensin influence crucial processes during interphase, and unforeseen aspects of mitosis. Each complex can perform several roles, and individual subunits can associate with different sets of proteins to achieve diverse functions, including the regulation of gene expression, DNA repair, cell-cycle checkpoints and centromere organization.

Adenosine Triphosphatases↗

PubMed1995

Identification of a germ-cell-specific transcriptional repressor in the promoter of Tctex-1.

The Tctex-1 gene family maps to the t complex of the mouse and consists of four copies on chromosome 17 in both wild-type and t-haplotypes. Tctex-1 mRNA is eightfold overexpressed in male and female germ cells in t-haplotype compound heterozygotes (tx/ty). In order to determine the cause of this aberrant expression and the role of this gene family in spermatogenesis and oogenesis it was subjected to extensive molecular analysis. We find that Tctex-1 protein is present in sperm tails and oocytes and that it is present at equal levels in wild-type and t-haplotype testis. Surprisingly, the excess message in t-haplotypes is not translated. Sequence analysis of the gene family reveals that one copy in t-haplotypes has a mutated start codon. This same copy is deleted for a protein-binding motif in its promoter. This motif, GIM (Germ cell Inhibitory Motif) has strong homology to the Xenopus AP-2-binding site but does not appear to be a binding site for mammalian AP-2. A factor(s) present in testis and ovary, but absent in other mouse tissues binds specifically to this site. Transfection assays using Tctex-1 promoter constructs suggest that GIM functions as a transcriptional repressor. The possible role of Tctex-1 in t complex transmission ratio distortion and sterility is discussed.

Animals↗

PubMed1993

Complex duplications in maize lines.

Rp1 is a disease resistance complex and is the terminal morphological marker on the short arm of maize chromosome 10. Several restriction fragment length polymorphisms (RFLPs), which map within 5 map units of Rp1, were examined to determine if they are also complex in structure. Two RFLP loci, which mapped distally to Rp1, BNL3.04 and PIO200075, existed in a single copy in all maize lines examined. These two loci cosegregated perfectly in 130 test cross progeny. Two RFLP loci that map proximally to Rp1 had unusual structures, which have not yet been reported for maize RFLPs; the loci were complex, with variable numbers of copies in different maize lines. One of the loci, NP1285, occasionally recombined in meiosis to yield changes in the number of copies of sequences homologous to the probe. The other proximal locus, detected by the probes NPI422, KSU3, and KSU4, was relatively stable in meiosis and no changes in the number of restriction fragments were observed. The similarity in map position between Rp1 and the complex RFLP loci indicate there may be genomic areas where variable numbers of repeated sequences are common. The structure of these complex loci may provide insight into the structure and evolution of Rp1.

Chromosome Mapping↗

PubMed1999

Posttranslational protein translocation across the membrane of the endoplasmic reticulum.

Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.

Animals↗

PubMed2003

Architecture of the ParF*ParG protein complex involved in prokaryotic DNA segregation.

The mechanism by which low copy number plasmids are segregated at cell division involves the concerted action of two plasmid-encoded proteins that assemble on a centromere-like site. This study explores the topology of the DNA segregation machinery specified by the parFG locus of TP228, a partition system which is phylogenetically distinct from more well-characterized archetypes. A variety of genetic, biochemical and biophysical strategies revealed that the ParG protein is dimeric. ParF, which is more closely related to the cell division regulator MinD than to the prototypical ParA partition protein of plasmid P1, is instead multimeric and its polymeric state appears to be modulated by ATP which correlates with the proposed ATP-binding activity of ParF. ParG interacts in a sequence-specific manner with the DNA region upstream of the parFG locus and this binding is modulated by ParF. Intriguingly, the ParF and ParG proteins form at least two types of discrete complex in the absence of this region suggesting that the assembly dynamics of these proteins onto DNA is intricate.

Adenosine Triphosphate↗

PubMed1996

The organization of endoplasmic reticulum export complexes.

Export of cargo from the ER occurs through the formation of 60-70nm COPII-coated vesicular carriers. We have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of ER export sites in vivo and in vitro. We find that ER buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. The basic organization of an export complex can be divided into an active COPII-containing budding zone on a single ER cisterna, which is adjacent to budding zones found on distantly connected ER cisternae. These budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain COPI coats involved in retrograde transport. Vesicles within these export complexes contain concentrated cargo molecules. The structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. We conclude that budding from the ER through recruitment of COPII is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by COPI.

Animals↗

PubMed1987

Visual memory and motor programmes: their use by idiot-savant artists and controls.

Artistically gifted children of normal intelligence and idiot-savant artists as well as two groups of IQ-matched controls were tested for their perception of and recognition memory for shapes. They were also tested for their capacity to graphically reproduce the shapes on sight (copying) or from memory. The material consisted of designs of two levels of complexity and structure. Results showed that while IQ was the determining factor in matching and recognition performance, copying and reproduction ability depended on artistic ability and was independent of level of intelligence. These results are interpreted in terms of access to an intelligence-independent system of graphic representations which can evoke appropriate motor programmes.

Adolescent↗

PubMed1997

Molecular cytogenetic characterization of cancer cell alterations.

Chromosomal abnormalities are the hallmark of cancer cells. Recurring and highly consistent structural and numerical alterations have been identified in a large number of leukemias, lymphomas, and solid tumors. The identification of recurrent genetic alterations and the isolation of molecular markers have clinical applications in the diagnosis and prognosis of neoplasia and in the detection of minimal residual disease that are essential for designing the most effective therapeutic approach. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are powerful techniques for detection of genomic alterations. The battery of FISH methods and DNA probes that are available can resolve virtually any chromosomal alterations regardless of their complexity. Combined chromosome banding, multifluor or spectral karyotype, and comparative genomic hybridization (CGH) allow identification of structural and numerical alterations on a global basis, mapping of the DNA copy number on the entire tumor genome, complete derivation of complex rearrangements, and localization of the breakpoints of translocations and deletions. Regions of recurrent alterations can be microdisected, amplified, microclone libraries constructed and probes localized on extended chromosomes or chromatin fibers for construction of high resolution physical maps that are critical for positional cloning and gene identification. In this review we attempted to cover the current trends in cancer molecular cytogenetics, and to outline the importance of molecular chromosome analysis in the understanding of oncogenesis and its clinical applications.

Chromosome Deletion↗

PubMed1998

Protein and lipid sorting between the endoplasmic reticulum and the Golgi complex.

Vesicular traffic within the early secretory pathway is mediated by COPI- and COPII-coated vesicles. While COPII-coated vesicles appear to be involved exclusively in the export of secretory proteins and lipids from the endoplasmic reticulum (ER), COPI-coated vesicles seem to function in both anterograde and retrograde transport between the ER-Golgi intermediate compartment (IC) and the Golgi as well as in intra-Golgi transport. Here, we focus on (i) the mechanisms how these transport carriers are formed from a given donor membrane; and (ii) the possible mechanisms involved in sorting of proteins and lipids into such transport vesicles.

Biological Transport↗

PubMed2005

Interactions of annexins with the mu subunits of the clathrin assembly proteins.

A number of biochemical and genetic studies have suggested that certain annexins play important roles in the endocytic pathway, possibly involving the generation, localization, or fusion of endocytic compartments. In a yeast two-hybrid screen for proteins that interact with the N-terminal domain of annexin A2 we identified the mu2 subunit of the clathrin assembly protein complex AP-2. The interaction depended upon two copies of a Yxx phi amino acid sequence motif (Y = tyrosine, x = variable residue, phi = bulky, hydrophobic residue) in the annexin that is also characteristic of the binding site for mu2 on the cytoplasmic domains of transmembrane receptors. The interaction between mu2 and full-length annexin A2 was demonstrated in vitro to be direct, to require calcium, and to be functional in the sense that annexin A2 was able to recruit the mu2 to immobilized lipids. Examination of other annexins and mu subunits demonstrated that annexin A2 also binds the mu1 subunit of the AP-1 complex, that annexin A6 binds mu1 and mu2, and that annexin A1 binds only mu1. We propose that annexins can "masquerade" as transmembrane receptors when they are attached to membranes in the presence of calcium and that they might therefore function to initiate calcium-regulated coated pit formation at the cell surface or on intracellular organelles.

Amino Acid Sequence↗

PubMed1993

Analysis of a nucleoprotein complex: the synaptosome of gamma delta resolvase.

The gamma delta resolvase protein is one of a large family of transposon-encoded site-specific recombinases. It performs recombination in a DNA-protein complex that contains 12 resolvase protomers and two copies of the 120-base pair DNA substrate, res (each with three binding sites for a resolvase dimer). A derivative of resolvase with altered DNA binding specificity was used to show that the role of resolvase at site I, which contains the crossover point, differs from its role at the other two binding sites. The resolvase dimers that initially bind to site I are the only ones that require the residue Ser10, essential for catalysis of DNA breakage. In addition, these site I-bound dimers do not use a specific interaction between dimers that is required elsewhere in the complex for synapsis of the res sites.

Base Sequence↗

PubMed1991

A 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase forms a stable complex with the catalytic subunit leading to markedly altered enzyme activity.

In an effort to clarify effects of specific protein-protein interactions on the properties of the dodecameric enzyme aspartate transcarbamoylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), we initiated studies of a simpler complex containing an intact catalytic trimer and three copies of a fragment from the regulatory chain. The partial regulatory chain was expressed as a soluble 9-kDa zinc-binding polypeptide comprising 11 amino acids encoded by the polylinker of pUC18 fused to the amino terminus of residues 84-153 of the regulatory chain; this polypeptide includes the zinc domain detected in crystallographic studies of the holoenzyme. In contrast to intact regulatory chains, the zinc-binding polypeptide is monomeric in solution because it lacks the second domain responsible for dimer formation and assembly of the dodecameric holoenzyme. The isolated 9-kDa protein forms a tight, zinc-dependent complex with catalytic trimer, as shown by the large shift in electrophoretic mobility of the trimer in nondenaturing polyacrylamide gels. Enzyme assays of the complex showed a hyperbolic dependence of initial velocity on aspartate concentration with Vmax and Km for aspartate approximately 50% lower than the values for free catalytic subunit. A mutant catalytic subunit containing the Lys-164----Glu substitution exhibited a striking increase in enzyme activity at low aspartate concentrations upon interaction with the zinc domain because of a large reduction in Km upon complex formation. These changes in functional properties indicate that the complex of the zinc domain and catalytic trimer is an analog of the high-affinity R ("relaxed") state of aspartate transcarbamoylase, as proposed previously for a transiently formed assembly intermediate composed of one catalytic and three regulatory subunits. Conformational changes at the active sites, resulting from binding the zinc-containing polypeptide chains, were detected by difference spectroscopy with trinitrophenylated catalytic trimers. Isolation of the zinc domain of aspartate transcarbamoylase provides a model protein for study of oligomer assembly, communication between dissimilar polypeptides, and metal-binding motifs in proteins.

Aspartate Carbamoyltransferase↗