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PubMed2003

Resistance profile of a hepatitis C virus RNA-dependent RNA polymerase benzothiadiazine inhibitor.

Recently, a benzo-1,2,4-thiadiazine antiviral agent (C(21)H(21)N(3)O(4)S; compound 4) was shown to be a potent, highly specific inhibitor of the primary catalytic enzyme of the hepatitis C virus (HCV) replicase complex. In this study, we selected for resistance to confirm the mechanism of action for compound 4 in HCV replicon cells. As expected, spontaneous mutations or fluidity in the HCV polymerase (NS5B) coding sequence occurred upon routine passage of the HCV replicon cells in the absence of compound 4. After 1 month of culture in the presence of 10 microM compound 4, or 20 times the 50% inhibitory concentration of the replicon, replicon cells were almost 20-fold less susceptible to compound 4. Twenty-one NS5B cDNA clones were generated from the resistant replicon cells. Five mutations in the 21 NS5B clones were present at frequencies higher than that of control replicon cells, and no clone contained more than a single mutation within the polymerase gene. RNA-dependent RNA polymerase studies using purified recombinant NS5B containing these single point mutations allowed the identification of residue 414 as sufficient for biochemical resistance to compound 4. Further, the contribution of this residue to confer cell-based resistance to compound 4 was validated using a stable recombinant mutant replicon cell line which harbors a methionine-to-threonine change at residue 414. The potential for additional mutations in other nonstructural genes of HCV to contribute to the resistance profile of compound 4 is discussed.

Benzothiadiazines↗

PubMed2003

Complementation of a deletion in the rubella virus p150 nonstructural protein by the viral capsid protein.

Rubella virus (RUB) replicons with an in-frame deletion of 507 nucleotides between two NotI sites in the P150 nonstructural protein (DeltaNotI) do not replicate (as detected by expression of a reporter gene encoded by the replicon) but can be amplified by wild-type helper virus (Tzeng et al., Virology 289:63-73, 2001). Surprisingly, virus with DeltaNotI was viable, and it was hypothesized that this was due to complementation of the NotI deletion by one of the virion structural protein genes. Introduction of the capsid (C) protein gene into DeltaNotI-containing replicons as an in-frame fusion with a reporter gene or cotransfection with both DeltaNotI replicons and RUB replicon or plasmid constructs containing the C gene resulted in replication of the DeltaNotI replicon, confirming the hypothesis that the C gene was the structural protein gene responsible for complementation and demonstrating that complementation could occur either in cis or in trans. Approximately the 5' one-third of the C gene was necessary for complementation. Mutations that prevented translation of the C protein while minimally disturbing the C gene sequence abrogated complementation, while synonymous codon mutations that changed the C gene sequence without affecting the amino acid sequence at the 5' end of the C gene had no effect on complementation, indicating that the C protein, not the C gene RNA, was the moiety responsible for complementation. Complementation occurred at a basic step in the virus replication cycle, because DeltaNotI replicons failed to accumulate detectable virus-specific RNA.

Amino Acid Sequence↗

PubMed2005

Nuclear matrix support of DNA replication.

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.

Animals↗

PubMed1993

Comparison of genetic maps for two Leptospira interrogans serovars provides evidence for two chromosomes and intraspecies heterogeneity.

Genetic maps were constructed for Leptospira interrogans serovars icterohaemorrhagiae and pomona. Previously we independently constructed physical maps of the genomes for these two serovars. The genomes of both serovars consist of a large replicon (4.4 to 4.6 Mb) and a small replicon (350 kb). Genes were localized on the physical maps by using Southern blot analysis with specific probes. Among the probes used were genes encoding a variety of essential enzymes and genes usually found near bacterial chromosomal replication origins. Most of the essential genes are on the larger replicon of each serovar. However, the smaller replicons of both serovars contain the asd gene. The asd gene encodes aspartate beta-semialdehyde dehydrogenase, an enzyme essential in amino acid and cell wall biosyntheses. The finding that both L. interrogans replicons contain essential genes suggests that both replicons are chromosomes. Comparison of the genetic maps of the larger replicons of the two serovars showed evidence of large rearrangements. These data show that there is considerable intraspecies heterogeneity in L. interrogans.

Amino Acids↗

PubMed2006

The C-terminal hydrophobic domain of hepatitis C virus RNA polymerase NS5B can be replaced with a heterologous domain of poliovirus protein 3A.

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.

Amino Acid Substitution↗

PubMed2003

Alphavirus cDNA-based expression vectors: effects of RNA transcription and nuclear export.

The construction of layered DNA-RNA replicons has facilitated and expanded the use of alphavirus vectors to vaccine development, construction of packaging cell lines and long-term heterologous gene expression. In these vector systems, the alphavirus replicon is under the control of a strong RNA polymerase II promoter and replicon RNA is transcribed from DNA before transport to the cytoplasm. Efficient RNA amplification catalyzed by the viral replicase results in high levels of mRNA and the recombinant protein. Recently, we developed a temperature-regulated Sindbis replicon-based DNA expression system characterized by a linear increase of expression upon decrease of the temperature from 37 degrees C to 29 degrees C. Modifications known to affect transcription and nuclear export of RNA led to a 5-fold increase in expression in BHK cells and up to over 80-fold increase in CHO cells and BF fibroblasts in transient transfection experiments. Furthermore, reducing cell proliferation resulted in a further 2- to 3-fold higher expression. While increased expression per cell was responsible for some of the enhanced production, it was primarily the number of expressing cells that made the difference in most cell lines. Further experiments indicated that a threshold amount of replicon RNA had to reach the cytoplasm in order for replication to occur. Thus, alterations that improve transcription, nuclear export and stability of the RNA had a significant impact on protein production in the pCytTS expression system and probably in other layered DNA-based viral vectors. Furthermore the results indicate that RNA replication is differentially regulated in DNA layered RNA replicons versus viral infection.

Active Transport, Cell Nucleus↗

PubMed2006

Negative regulation of intracellular hepatitis C virus replication by interferon regulatory factor 3.

BACKGROUND: Interferon regulatory factor (IRF)-3 plays an important role in initiating cellular interferon-stimulated gene-mediated antiviral responses. In the present study, we evaluated the effects of IRF-3 expression and activation on intracellular hepatitis C virus (HCV) replication using an HCV replicon system. METHODS: An HCV replicon was constructed that expressed a neomycin-selectable chimeric firefly luciferase reporter protein. A small interfering (si) RNA oligonucleotide directed against IRF-3 mRNA was designed and synthesized. A eukaryote expression plasmid vector was constructed that expressed IRF-3 mRNA under control of the cytomegalovirus early promoter/enhancer. To evaluate transcriptional activity of the interferon-stimulated genes, a reporter vector was used that expressed firefly luciferase under control of the interferon-stimulated response element (ISRE). RESULTS: The baseline expression of IRF-3 did not significantly differ between cells with and without expression of the replicon. Transfection of an IRF-3 expression plasmid into the cells raised the ISRE-luciferase activities. The increase of ISRE activity was significantly more potent in the replicon-expressing cells than in cells without replicon expression. Concomitantly, the overexpression of IRF-3 suppressed HCV replication levels. In contrast, siRNA knockdown of IRF-3 suppressed ISRE activity by 38% +/- 2%. Interestingly, the suppression of IRF-3 resulted in a significant increase of HCV replication, by up to twofold, depending on the IRF-3 suppression levels. CONCLUSIONS: IRF-3 negatively regulated intracellular HCV replication, and was partially activated in cells that expressed the HCV replicon. Thus, IRF-3 is a key molecule controlling HCV replication through modulation of host interferon gene responses.

Blotting, Western↗

PubMed1984

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment.

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [3H]TdR for 30 min, and chased in medium containing 1 X 10(-6) M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9 - 12 X 10(6) D single-stranded (ss) DNA. When the cells were chased in medium with 1 X 10(-5) M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 X 10(-4) M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.

DNA Replication↗

PubMed2004

Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species.

Bacillus megaterium QM B1551 contains seven plasmids. Two are small rolling circle plasmids and five are theta-replicating plasmids with cross-hybridizing replicons that define a new family of very homologous yet compatible theta replicons. Previous sequencing of several of the plasmids has shown genes with high similarity to those on the genomes and plasmids of other Gram-positive bacteria. To test the possible distribution of these plasmids, nine other B. megaterium strains and 20 other Bacillus or related species were tested for the presence of similar replicons, and specific flanking DNA by both hybridization and PCR. The theta replicons were widespread among the B. megaterium strains, and two had one or more of the rolling circle plasmids, but none of the plasmid replicon regions were observed in the other Bacillus or related species. It appears from the data that even though some plasmids carry genes suggesting horizontal transfer, their replicons seem to be unique to B. megaterium, or rarely present in related species.

Amino Acid Sequence↗

PubMed1982

Molecular homology and incompatibility in the IncFI plasmid Group.

The usual grounds for the inclusion of a plasmid in a particular incompatibility group are its mutual incompatibility with a type plasmid of that group, and, in some cases, the demonstration of shared regions of specific homology, presumed to be related to DNA replication. We have found that some plasmids classified as IncFI on genetical grounds share no homology with the previously described incompatibility regions of F on the basis of hybridization of specific radioactive probes to restriction enzyme digests of DNA from these plasmids. Others show homology with some or all of the regions of the F plasmid that can express incompatibility. The incompatibility behaviour of these plasmids has been examined to determine the relationship between the possession of regions of homology and the expression of incompatibility. Three plasmids, ColV3-K30, pHH507 and Entp307, show homology only with the secondary replicon of F and appear to use sequences homologous with the secondary F replicon in their replication. The results are consistent with the propositions that some contemporary IncFI plasmids arose by the integration of several replicons, and, in general, the replicon not being used for replicon expresses its incompatibility, as does the replicon being used for replication. We conclude that incompatibility of two plasmids with F does not necessarily demonstrate relatedness of the plasmids to each other, and that inclusion within the IncFI group can result from the possession of any of several combinations of inc sequences.

Base Sequence↗

PubMed2005

Hairpin telomeres and genome plasticity in Borrelia: all mixed up in the end.

Spirochetes of the genus Borrelia have a highly unusual genome structure composed of over 20 replicons. Most of these replicons are linear and terminated by covalently closed hairpin ends or telomeres. Moreover, the linear replicons are affected by extensive DNA rearrangements, including telomere exchanges, DNA duplications, and harbour a large number of pseudogenes. The mechanism for the unusual genome plasticity in the linear replicons has remained elusive. The enzymatic machinery (the telomere resolvase ResT) responsible for generating the hairpin ends from replicative intermediates has recently been shown to also perform a reverse reaction that fuses telomeres on unrelated replicons. Infrequent stabilization of such fusion events over evolutionary time provides the first proposed biochemical mechanism for the DNA rearrangements that are so prominent in the linear replicons of B. burgdorferi.

Bacterial Proteins↗

PubMed2005

Molecular characterization of plasmid pBM300 from Bacillus megaterium QM B1551.

Strain QM B1551 of Bacillus megaterium contains seven compatible plasmids: two small rolling circle plasmids and five theta-replicating plasmids with cross-hybridizing replicons. To expand our understanding of these plasmids, the replicon region (6.7 kb) from pBM300 was cloned, sequenced, and functionally characterized. Sequence analysis showed that the replication protein (RepM300) was highly homologous to two other plasmid Rep proteins of the same strain but to no other known proteins. Furthermore, the location of the replication origin was within the RepM300 coding region, and the origin contained three 12-base direct repeats. Deletion analysis of the replicon confirmed the role of the Rep protein and showed that open reading frame 2 (ORF2) was required for stability. However, the protein encoded by ORF2 is entirely different from the replicon stability proteins encoded by the other two replicons. The entire plasmid was isolated from the plasmid array by integrating a spectinomycin resistance gene and transforming a plasmidless strain, PV361. Complete sequencing showed that pBM300 was 26,300 bp long, had a G+C content of 35.2%, and contained 20 ORFs, two of which encoded proteins that had no similarity to other proteins in the database. The proteins encoded by the plasmid ORFs had similarity to proteins for mobilization and transfer, an integrase, a rifampin resistance protein, a cell wall hydrolase, glutathione synthase, and a biotin carboxylase. The similarities were to several gram-positive genera and a few gram-negative genera and archaea. oriT and ssoT-like regions were detected near two mob genes. These results suggest that pBM300 is a mobilizable hybrid plasmid that confers increased metabolic and germination ability on its host. Its replicon also helps define a new plasmid family.

Bacillus megaterium↗

PubMed1996

Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population. Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing. The relative contribution of each system to stabilization depends on plasmid copy number and the particular E. coli host.

Bacterial Proteins↗

PubMed2006

Relationships between hepatitis C virus replication and CXCL-8 production in vitro.

The chemokine CXCL-8 (interleukin-8) is induced by many viruses, including hepatitis C virus (HCV). In the current study, we examined CXCL-8 levels in the context of acute and chronic HCV replication in vitro. Two different small interfering RNAs were used to silence CXCL-8 mRNA and protein expression in Huh7 and BB7 replicon cells. HCV RNA synthesis in BB7 cells was inhibited by CXCL-8 knockdown. Furthermore, antibody neutralization of endogenous CXCL-8 activity inhibited HCV replication, while addition of recombinant human CXCL-8 stimulated NS5A protein expression. Moreover, CXCL-8 protein levels correlated positively with HCV RNA levels in four independent subgenomic and genomic replicon lines (R = 0.41, P = 0.0013). However, CXCL-8 mRNA levels correlated inversely with CXCL-8 protein and HCV RNA levels in all replicon lines and in Huh7 cells. Transient replication assays with strongly permissive and weakly permissive Huh7 cells and three independent subgenomic replicons with various replicative capacities revealed that CXCL-8 protein levels were higher in weakly than in strongly permissive cells. The JFH-1 subgenomic replicon, which replicated to high levels in both strongly and weakly permissive Huh7 cells, induced CXCL-8 protein to high levels in both cell types. The data indicate that in the replicon system, CXCL-8 protein levels are positively associated with chronic HCV replication and that CXCL-8 removal inhibits HCV replication. During acute HCV replication, CXCL-8 production may be inhibitory to viruses with low replicative capacity. The data underscore the complex regulation of CXCL-8 mRNA and protein expression and further suggest that in addition to contributing to HCV pathology via proinflammatory actions, CXCL-8 may have opposing antiviral and proviral effects depending on the level of HCV replication, the cellular context, and whether the infection is acute or chronic.

Cell Line, Tumor↗

PubMed1999

Characterization of the oriI and oriII origins of replication in phage-plasmid P4.

In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the alpha and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the alpha gene, with the internal ori2 site, and the crr region. The P4 alpha protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the alpha protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the alpha protein did not bind ori2. Thus, the alpha protein appears to act differently at the two origins of replication.

Bacterial Proteins↗

PubMed2006

Mechanistic link between the anti-HCV effect of interferon gamma and control of viral replication by a Ras-MAPK signaling cascade.

Interferon-gamma (IFN-gamma) exerts potent antiviral activity in the hepatitis C virus (HCV) replicon systems. However, the mechanisms underlying the direct antiviral effect have not been determined. We found that the type II transcriptional response to IFN-gamma could be suppressed by inhibition of MEK1/2 kinase activity by MEK1/2 inhibitor U0126 in the hepatoma cell line Huh-7. Using a bicistronic HCV replicon system expressing a luciferase reporter gene in Huh-7 cells (RLuc-replicon), we showed that inhibition of MEK1/2 kinase activity is sufficient to counteract the antiviral activity of IFN-gamma. Expression of a constitutive active form of Ras inhibited the luciferase activity of RLuc-replicon, whereas a dominant-negative mutant of Ras enhanced the reporter activity, indicating that the Ras-MAPK pathway has a role in limiting replication of the viral RNA. Consistent with the involvement of the Ras-MAPK pathway, treatment with epidermal growth factor suppressed HCV protein expression in the RLuc-replicon cells, an effect that could be abolished by U0126. Inhibition of MEK1/2 kinase activity correlated with reduced phosphorylation of the HCV NS5A protein and enhanced RLuc-replicon luciferase reporter activity, in line with recent reports that phosphorylation of NS5A negatively modulates HCV RNA replication. Finally, genetic deletion analysis in yeast supported the role of a MEK-like kinase(s) in the regulation of NS5A phosphorylation. In conclusion, the direct anti-HCV effect of IFN-gamma in cell culture is, at least in part, mediated through the Ras-MAPK signaling pathway, which possibly involves a direct or indirect modulation of NS5A protein phosphorylation.

Antiviral Agents↗

PubMed1977

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro.

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of 3H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which last 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5-fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 mum/h (38kb). The mean distance between the middle points of adjacent labeled regions was 70 mgm (210 kb). The size of most replicons ranged from 40 to 120 mum. - These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. - On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.

Animals↗

PubMed1981

Studies of mammalian chromosome replication. II. Evidence for the existence of defined chromosome replicating units.

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G1/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these later intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 micrometers in all S phase cells except those at G1/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units of chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.

Animals↗