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PubMed2001

Inheritance of the replication complex: a unique or common phenomenon in the control of DNA replication?

Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage lambda demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for lambda plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.

Bacteriophage lambda↗

PubMed2000

RNA secondary structures of the bacteriophage phi6 packaging regions.

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

Bacteriophage phi 6↗

PubMed1997

Inborn errors of the Krebs cycle: a group of unusual mitochondrial diseases in human.

Krebs cycle disorders constitute a group of rare human diseases which present an amazing complexity considering our current knowledge on the Krebs cycle function and biogenesis. Acting as a turntable of cell metabolism, it is ubiquitously distributed in the organism and its enzyme components encoded by supposedly typical house-keeping genes. However, the investigation of patients presenting specific defects of Krebs cycle enzymes, resulting from deleterious mutations of the considered genes, leads to reconsider this simple envision by revealing organ-specific impairments, mostly affecting neuromuscular system. This often leaves aside organs the metabolism of which strongly depends on mitochondrial energy metabolism as well, such as heart, kidney or liver. Additionally, in some patients, a complex pattern of tissue-specific enzyme defect was also observed. The lack of functional additional copies of Krebs cycle genes suggests that the complex expression pattern should be ascribed to tissue-specific regulations of transcriptional and/or translational activities, together with a variable cell adaptability to Krebs cycle functional defects.

Adolescent↗

PubMed2004

The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex.

The Tat system transports folded proteins across bacterial plasma and plant thylakoid membranes. To date, three key Tat subunits have been identified and mechanistic studies indicate the presence of two types of complex: a TatBC-containing substrate-binding unit and a separate TatA complex. Here, we used blue-native gel electrophoresis and affinity purification to study the nature of these complexes in Escherichia coli. Analysis of solubilized membrane shows that the bulk of TatB and essentially all of the TatC is found in a single 370kDa TatABC complex. TatABC was purified to homogeneity using an affinity tag on TatC and this complex runs apparently as an identical band. We conclude that this is the primary core complex, predicted to contain six or seven copies of TatBC together with a similar number of TatA subunits. However, the data indicate the presence of an additional form of Tat complex containing TatA and TatB, but not TatC; we speculate that this may be an assembly or disassembly intermediate of the translocator. The vast majority of TatA is found in separate complexes that migrate in blue-native gels as a striking ladder of bands with sizes ranging from under 100 kDa to over 500 kDa. Further analysis shows that the bands differ by an average of 34 kDa, indicating that TatA complexes are built largely, but possibly not exclusively, from modules of three or four TatA molecules. The range and nature of these complexes are similar in a TatC mutant that is totally inactive, indicating that the ladder of bands does not stem from ongoing translocation activity, and we show that purified TatA can self-assemble in vitro to form similar complexes. This spectrum of TatA complexes may provide the flexibility required to generate a translocon capable of transporting substrates of varying sizes across the plasma membrane in a folded state.

Arginine↗

PubMed2003

Genetic and cytogenetic analyses of breast cancer yield different perspectives of a complex disease.

Genomic instability in breast cancer results in low-level changes in DNA copy number, a significant but poorly understood mechanism underlying the genetic heterogeneity of this disorder. Two different approaches, loss of heterozygosity (LOH) and comparative genomic hybridization (CGH), have been used to probe the genetics of breast cancer evolution. LOH is a locus specific method that detects the variation in the parental origin of DNA, but is not quantitative. CGH provides a genome-wide accounting of the magnitude of DNA copy number changes, but not parental origin. Both methods have identified complex and heterogeneous patterns of DNA losses, duplications, and amplifications during breast cancer evolution. LOH and CGH technologies interrogate very distinct mechanisms driving breast tumor evolution, yet are seldom used in parallel to profile specimens. Thus, the relative significance of genetic versus numerical variations of DNA in breast cancer evolution remains undefined. This review will attempt to summarize some of the successes of these investigations, highlight some complex and confounding observations emerging from these studies, and discuss the potential of these studies to improve our understanding of breast cancer biology and treatment.

Breast Neoplasms↗

PubMed1975

Studies on Macronuclear DNA from Paramecium aurelia.

Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction. DNA molecules as long as 105 microns and as short as 0.2 microns were observed. It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase. Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E. coli DNA was determined. This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA. Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E. coli DNA which corresponded to 3 X 10(10) daltons. There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus. Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA. Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA. Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome. These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA.

Animals↗

PubMed2004

Structural insight into the protein translocation channel.

A structurally conserved protein translocation channel is formed by the heterotrimeric Sec61 complex in eukaryotes, and SecY complex in archaea and bacteria. Electron microscopy studies suggest that the channel may function as an oligomeric assembly of Sec61 or SecY complexes. Remarkably, the recently determined X-ray structure of an archaeal SecY complex indicates that the pore is located at the center of a single molecule of the complex. This structure suggests how the pore opens perpendicular to the plane of the membrane to allow the passage of newly synthesized secretory proteins across the membrane and opens laterally to allow transmembrane segments of nascent membrane proteins to enter the lipid bilayer. The electron microscopy and X-ray results together suggest that only one copy of the SecY or Sec61 complex within an oligomer translocates a polypeptide chain at any given time.

Bacterial Proteins↗

PubMed2003

Identification of a third sulfate activation system in Sinorhizobium sp. strain BR816: the CysDN sulfate activation complex.

Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3'-phosphoadenosine-5'-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the "household" CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.

Bacterial Proteins↗

PubMed1997

Heterochromatic Stellate gene cluster in Drosophila melanogaster: structure and molecular evolution.

The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 beta-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (approximately 14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.

Amino Acid Sequence↗

PubMed1994

Characterization of two distinct gene transcripts for ribosomal protein L21 from pathogenic and nonpathogenic strains of Entamoeba histolytica.

A second gene (rp-L21) copy, clone g34, coding for ribosomal (r-) protein L21, was isolated from the pathogenic (P) strain HM-1:IMSS cl6 of the intestinal parasite Entamoeba histolytica (Eh). The gene was compared to the previously isolated copy, gLE3 [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], with respect to its primary structure, mRNA levels and binding to the r-complex during translation. Unlike the gLE3 gene copy [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], g34 was found not to be physically connected to an actin gene copy. Homologous copies of the two rp-L21 genes were also characterized from the nonpathogenic (NP) strain SAW1734R clAR, as well as from its P derivative. Sequence comparison of the coding regions of the two rp-L21 revealed almost full identity. Significant differences were found, however, within their 3' and 5' flanking regions. Using the 3' rapid amplification of cDNA ends (3' RACE) method [Frohman et al., Proc. Natl. Acad. Sci. USA 85 (1988) 8998-9002], as well as Northern and slot blot hybridizations, it was demonstrated that both rp-L21 mRNAs are found in similar amounts. However, as was shown by differential hybridization, the relative binding of each transcript to the r-complex varied somewhat between P and NP strains. This finding suggests that the control of expression of rp-L21 in Eh may involve regulation at the post-transcriptional level.

Amino Acid Sequence↗

PubMed1989

Complex formation between influenza virus polymerase proteins expressed in Xenopus oocytes.

All three influenza virus polymerase (P) proteins were expressed in Xenopus oocytes from microinjected in vitro transcribed mRNA analogs, with yields of up to 100 ng per oocyte. To examine the functional state of the Xenopus-expressed P proteins, the polypeptides were tested for their ability to form stable complexes with each other. As seen in virus-infected cells, all three P proteins associated into an immunoprecipitable complex, suggesting that the system has considerable promise for the reconstruction of an active influenza RNA polymerase. Examination of the ability of paired combinations of the P proteins to associate indicated that PB1 contained independent binding sites for PB2 and PA, and so probably formed the backbone of the complex. Sedimentation analysis of free and complexed P proteins indicated that PB1 and PB2 did not exist as free monomers, and that similarly, complexes of all three P proteins did not simply consist of one copy of each protein. The heterodisperse sedimentation rate seen for complexes of all three P proteins did not appear to result from their binding to RNA, suggesting the incorporation of additional polypeptides in the polymerase complex.

Animals↗

PubMed2000

Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum.

Mnn9p is a component of two distinct multiprotein complexes in the Saccharomyces cerevisiae cis-Golgi that have both been shown to have alpha-1,6-mannosyltransferase activity in vitro. In one of these complexes, Mnn9p associates with four other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the other complex consists of Mnn9p and Van1p. Members of the Mnn9p-containing complexes were incorporated into COPII vesicles made in vitro from endoplasmic reticulum (ER) membranes isolated from cycloheximide-treated cells. This behavior is consistent with an active Golgi to ER recycling process. To examine this path in vivo, we monitored retrograde transport of subunits of the complex in cells blocked in anterograde transport from the ER. In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis. Conversely, when retrograde transport was blocked in vivo, subunits of the mannosyltransferase complex accumulated in the vacuole. Packaging of Mnn9p in COPI-coated vesicles from purified Golgi membranes was also investigated using a coatomer-dependent vesicle budding assay. Gradient fractionation experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles. These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golgi.

Cycloheximide↗

PubMed1975

Messenger RNA complexity in Drosophila melanogaster.

Complementary DNA was synthesized as a copy of polyadenylated RNA from the cytoplasm of Drosophila cultured cells. The kinetics of hybridization of cDNA with the RNA used as template revealed a complex distribution of frequencies in the population of polyadenylated RNA. Computer simulation suggested three frequency classes containing about 4, 190, and 6700 different RNA molecules of mean molecular weight 4 x 10-5. About 15% of this complementary DNA reacted with repetitive sequences of Drosophila DNA. The most frequent polyadenylated RNA is preferentially enriched in its content of repetitive sequences. Comparative experiments using cDNA synthesized as a complement of larval polyadenylated RNA demonstrated some stage specific changes in the population of polyadenylated RNA.

Cells, Cultured↗

PubMed2000

Isolation of a new retrotransposon-like DNA sequence and its use in analysis of diversity within the Oryza officinalis complex.

To better understand the genetic diversity of the wild relatives of rice (Oryza sativa L.) in the O. officinalis species complex repetitive DNA markers were obtained from the diploid species of this complex. One cloned sequence from O. eichingeri gave intense hybridization signals with all species of the O. officinalis complex. This 242 bp clone, named pOe.49, has a copy number from 0.9 to 4.0 x 10(4) in diploid species of this complex. Analysis of the primary structure and database searches revealed homology of pOe.49 to a number of sequences representing part of the integrase coding domain of retroviruses and gypsy-like retrotransposons. Sequencing of specific PCR products confirmed that pOe.49 is part of a gypsy-like retrotransposon. RFLP analysis was used to study the genomic organisation of pOe.49 among 30 accessions of the O. officinalis complex using 10 restriction enzymes. Diversity analysis based on 120 polymorphic fragments obtained from the RFLP assay grouped the O. officinalis complex accessions by genome, species and eco-geographic groups. The results suggest that, with further characterization, this retrotransposon-like DNA sequence may be useful for phylogenetic analysis of species in the O. officinalis complex.

Amino Acid Sequence↗

PubMed1999

Transformation of the nematode-trapping fungus Arthrobotrys oligospora.

The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters. Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl2 treatment. Transformation frequencies varied between 1-6 transformants per microgram DNA. Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations. The addition of restriction enzymes during transformations increased the frequency of single copy integrations. Co-transformation, using the E. coli uidA gene encoding the beta-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.

Animals↗

PubMed1993

Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae.

The chromatin structure of yeast ribosomal DNA was analyzed in vivo by crosslinking intact cells with psoralen. We found that in exponentially growing cultures the regions coding for the 35S rRNA precursor fall into two distinct classes. One class was highly accessible to psoralen and associated with nascent RNAs, characteristic for transcriptionally active rRNA genes devoid of nucleosomes, whereas the other class showed a crosslinking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rRNA gene copies. By crosslinking the same strain growing in complex or minimal medium, we have shown that yeast cells can modulate the proportion of active (non-nucleosomal) and inactive (nucleosomal) rRNA gene copies in response to variations in environmental conditions which suggests that yeast can regulate rRNA synthesis by varying the number of active gene copies, in contrast to the vertebrate cells studied so far. Whereas intergenic spacers flanking inactive rRNA gene copies are packaged in a regular nucleosomal array, spacers flanking active genes show an unusual crosslinking pattern suggesting a complex interaction of regulatory factors and histones with DNA.

Chromatin↗

PubMed2004

The entire Nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis.

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of approximately 30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.

Animals↗

PubMed1998

The pyruvate dehydrogenase multi-enzyme complex from Gram-negative bacteria.

Pyruvate dehydrogenase multi-enzyme complexes from Gram-negative bacteria consists of three enzymes, pyruvate dehydrogenase/decarboxylase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3). The acetyltransferase harbors all properties required for multi-enzyme catalysis: it forms a large core of 24 subunits, it contains multiple binding sites for the E1p and E3 components, the acetyltransferase catalytic site and mobile substrate carrying lipoyl domains that visit the active sites. Today, the Azotobacter vinelandii complex is the best understood oxo acid dehydrogenase complex with respect to structural details. A description of multi-enzyme catalysis starts with the structural and catalytic properties of the individual components of the complex. Integration of the individual properties is obtained by a description of how the many copies of the individual enzymes are arranged in the complex and how the lipoyl domains couple the activities of the respective active sites by way of flexible linkers. These latter aspects are the most difficult to study and future research need to be aimed at these properties.

Acetyltransferases↗