scholarly journals DiaA, a Novel DnaA-binding Protein, Ensures the Timely Initiation ofEscherichia coliChromosome Replication

2004 ◽  
Vol 279 (44) ◽  
pp. 45546-45555 ◽  
Author(s):  
Takuma Ishida ◽  
Nobuyoshi Akimitsu ◽  
Tamami Kashioka ◽  
Masakazu Hatano ◽  
Toshio Kubota ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Replication Initiation ◽  
Temperature Sensitive ◽  
Minichromosome Maintenance ◽  
Chromosomal Replication ◽  
Timely Initiation ◽  
Initiation Of Replication ◽  
Direct Binding ◽  
Cold Sensitive

The DnaA protein is the initiator ofEscherichia colichromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitivednaAmutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. AdiaA::Tn5mutation suppresses the cold-sensitive growth of an overinitiation typednaAmutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in thediaAmutant cells as well as wild-type cells with pBR322 expressing thediaAgene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in anin vitrosystem especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication.

scholarly journals Insulin biosynthesis in the bullhead, Ictalurus nebulosus, and the effect of temperature

1973 ◽  
Vol 134 (3) ◽  
pp. 753-761 ◽  
Author(s):  
Margaret L. Moule ◽  
Cecil C. Yip
Keyword(s):  
Qualitative Difference ◽  
Gel Filtration ◽  
Effect Of Temperature ◽  
Insulin Biosynthesis ◽  
Temperature Sensitive ◽  
Bicarbonate Buffer ◽  
Control Tissue ◽  
Ictalurus Nebulosus ◽  
Lower Temperature

Insulin biosynthesis in the brown bullhead, Ictalurus nebulosus (Le Sueur), was studied by measuring the incorporation in vitro of [3H]leucine into proteins of the principal islet. The tissue was incubated for 6–15h in Krebs–Ringer bicarbonate buffer with [3H]leucine, supplemented with amino acids and glucose. Proteins, precipitated with trichloroacetic acid and extracted with acid ethanol, were separated by gel-filtration on Biogel P-30 in 3m-acetic acid. Three major components were found after incubation of the islets at 22°C. On the basis of the results of sulphitolysis, biological activity and the demonstrated precursor–product relationship, components I and II were identified as proinsulin and insulin respectively. The third component was not identified. At 12°C, [3H]leucine was incorporated only into proinsulin. No radioactivity was found in insulin or the unidentified component III at 12°C as was found after incubation at 22°C. When the temperature was lowered from 22° to 12°C after 3h of a 15h incubation, decreased conversion of proinsulin into insulin resulted at the lower temperature compared with the control tissue maintained at 22°C. When the temperature was raised from 12° to 22°C at 3h of a 15h incubation, conversion of proinsulin into insulin occurred. No conversion occurred in the control tissue with the temperature maintained at 12°C. No qualitative difference in the incorporation of [3H]leucine into proinsulin and its conversion into insulin at 12° and 22°C could be demonstrated between islet tissue from fish acclimated to less than 12°C or to 22°C. The results suggest that the enzyme(s) responsible for converting proinsulin into insulin in the bullhead may be temperature sensitive with low activity at 12°C.

scholarly journals Association of Fission Yeast Orp1 and Mcm6 Proteins with Chromosomal Replication Origins

1999 ◽  
Vol 19 (10) ◽  
pp. 7228-7236 ◽  
Author(s):  
Yuya Ogawa ◽  
Tatsuro Takahashi ◽  
Hisao Masukata
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Chromatin Immunoprecipitation ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Minichromosome Maintenance ◽  
Chromatin Immunoprecipitation Assay ◽  
Chromosomal Replication ◽  
Initiation Of Replication ◽  
Mcm Proteins

ABSTRACT We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by thenda1 +/cdc19+ andmis5+ genes, respectively, were associated with chromatin DNA only during the G1 and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G1 and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 andars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G1 and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.

Thermal effects on neuronal activity in suprachiasmatic nuclei of hibernators and nonhibernators

1994 ◽  
Vol 266 (4) ◽  
pp. R1259-R1266 ◽  
Author(s):  
J. D. Miller ◽  
V. H. Cao ◽  
H. C. Heller
Keyword(s):  
Thermal Sensitivity ◽  
Large Population ◽  
Ground Squirrels ◽  
Neuronal Firing ◽  
Temperature Sensitive ◽  
Suprachiasmatic Nuclei ◽  
Circadian Fluctuation ◽  
Cold Sensitive ◽  
Deep Hibernation

The temperature sensitivity of neuronal firing rates in the suprachiasmatic nuclei (SCN) of the hypothalami of rats and ground squirrels was studied in vitro. SCN from euthermic squirrels were studied during the hibernation season (winter) and during the summer. SCN from hibernating squirrels were also studied. Most properties of SCN cells from hibernators and nonhibernators were similar. Warm- and cold-sensitive neurons were observed in all groups, but cold-sensitive neurons were more common in SCN from hibernating squirrels. No evidence for temperature compensation of firing rate was accumulated; no cell was observed to fire below 16.6 degrees C. If the persistence of circadian rhythmicity is a function of action potential-dependent neurotransmission from the SCN, these results suggest that deep hibernation (5-17 degrees C) should be characterized by an absence of circadian fluctuation in temperature. Two possible adaptations for the shallow torpor seen at somewhat higher temperatures were observed in the SCN: 1) a relatively large population of cold-sensitive neurons and 2) a population of neurons with very high activation energies. Activation energy analysis suggested that most of the temperature-sensitive properties of these cells could be explained in terms of the thermal sensitivity of the sodium channel.

A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids

1986 ◽  
Vol 6 (11) ◽  
pp. 4053-4059
Author(s):  
Y Kikuchi ◽  
A Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Complementation Group ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Initiation Of Replication ◽  
Low Levels ◽  
Stable Maintenance

We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

scholarly journals A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids.

1986 ◽  
Vol 6 (11) ◽  
pp. 4053-4059 ◽  
Author(s):  
Y Kikuchi ◽  
A Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Complementation Group ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Initiation Of Replication ◽  
Low Levels ◽  
Stable Maintenance

We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

scholarly journals Condensin locates at transcriptional termination sites in mitosis, possibly releasing mitotic transcripts

Open Biology ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 190125 ◽  
Author(s):  
Norihiko Nakazawa ◽  
Orie Arakawa ◽  
Mitsuhiro Yanagida
Keyword(s):  
Transcriptional Activation ◽  
Temperature Sensitive ◽  
Growth Defects ◽  
Transcriptional Termination ◽  
Genome Wide ◽  
Transcription Sites ◽  
Cold Sensitive ◽  
Inducible Genes ◽  
Mitotic Chromosome Condensation

Condensin is an essential component of chromosome dynamics, including mitotic chromosome condensation and segregation, DNA repair, and development. Genome-wide localization of condensin is known to correlate with transcriptional activity. The functional relationship between condensin accumulation and transcription sites remains unclear, however. By constructing the auxin-inducible degron strain of condensin, herein we demonstrate that condensin does not affect transcription itself. Instead, RNA processing at transcriptional termination appears to define condensin accumulation sites during mitosis, in the fission yeast Schizosaccharomyces pombe . Combining the auxin-degron strain with the nda3 β-tubulin cold-sensitive (cs) mutant enabled us to inactivate condensin in mitotically arrested cells, without releasing the cells into anaphase. Transcriptional activation and termination were not affected by condensin's degron-mediated depletion, at heat-shock inducible genes or mitotically activated genes. On the other hand, condensin accumulation sites shifted approximately 500 bp downstream in the auxin-degron of 5′-3′ exoribonuclease Dhp1, in which transcripts became aberrantly elongated, suggesting that condensin accumulates at transcriptionally terminated DNA regions. Growth defects in mutant strains of 3′-processing ribonuclease and polyA cleavage factors were additive in condensin temperature-sensitive (ts) mutants. Considering condensin's in vitro activity to form double-stranded DNAs from unwound, single-stranded DNAs or DNA-RNA hybrids, condensin-mediated processing of mitotic transcripts at the 3′-end may be a prerequisite for faithful chromosome segregation.

scholarly journals Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication

2006 ◽  
Vol 87 (11) ◽  
pp. 3373-3377 ◽  
Author(s):  
Tao Deng ◽  
Jane L. Sharps ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Replication Initiation ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Initiation Of Replication ◽  
Rna Genome ◽  
Transcription And Replication

Both transcription and replication of the influenza virus RNA genome are catalysed by a virus-specific RNA polymerase. Recently, an in vitro assay, based on the synthesis of pppApG, for the initiation of replication by recombinant RNA polymerase in the absence of added primer was described. Here, these findings are extended to show that adenosine, AMP and ADP can each substitute for ATP in reactions catalysed by either recombinant ribonucleoprotein or RNA polymerase complexes with either model virion RNA (vRNA) or cRNA promoters. The use of either adenosine or AMP, rather than ATP, provides a convenient, sensitive and easy assay of replication initiation. Moreover, no pppApG was detected when a PB1–PA dimer, rather than the trimeric polymerase, was used to catalyse synthesis, contrasting with a previous report using baculovirus-expressed influenza RNA polymerase. Overall, it is suggested that the heterotrimeric polymerase is essential for the initiation of replication.

scholarly journals Adaptor Complex-independent Clathrin Function in Yeast

1999 ◽  
Vol 10 (11) ◽  
pp. 3643-3659 ◽  
Author(s):  
Bonny G. Yeung ◽  
Huan L. Phan ◽  
Gregory S. Payne
Keyword(s):  
Gel Filtration ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
Binding Assays ◽  
Genes Encoding ◽  
Clathrin Adaptor ◽  
In Cells

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes inSaccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and α-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or α-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the β subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

scholarly journals Topoisomerase I Associates Specifically with Simian Virus 40 Large-T-Antigen Double Hexamer–Origin Complexes

2000 ◽  
Vol 74 (11) ◽  
pp. 5224-5232 ◽  
Author(s):  
Dahai Gai ◽  
Rupa Roy ◽  
Chunxiao Wu ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Replication Initiation ◽  
Initiation Complex ◽  
Initiation Of Replication ◽  
Sv40 Origin

ABSTRACT Topoisomerase I (topo I) is required for releasing torsional stress during simian virus 40 (SV40) DNA replication. Recently, it has been demonstrated that topo I participates in initiation of replication as well as in elongation. Although T antigen and topo I can bind to one another in vitro, there is no direct evidence that topo I is a component of the replication initiation complex. We demonstrate in this report that topo I associates with T-antigen double hexamers bound to SV40 origin DNA (TDH) but not to single hexamers. This association has the same nucleotide and DNA requirements as those for the formation of double hexamers on DNA. Interestingly, topo I prefers to bind to fully formed TDH complexes over other oligomerized forms of T antigen associated with the origin. High ratios of topo I to origin DNA destabilize TDH. The partial unwinding of a small-circular-DNA substrate is dependent on the presence of both T antigen and topo I but is inhibited at high topo I concentrations. Competition experiments with a topo I-binding fragment of T antigen indicate that an interaction between T antigen and topo I occurs during the unwinding reaction. We propose that topo I is recruited to the initiation complex after the assembly of TDH and before unwinding to facilitate DNA replication.

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