Control Of Chromosome Replication Research Articles

XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition

Persistent R-loops (RNA–DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5′-3′-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2’s association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.

Abstract P2-06-01: Intrinsic transcriptomic differences of luminal progenitors and mature luminal cells of the normal breast

Abstract Epithelial cells in the normal breast are organized hierarchically into stem, luminal progenitor (LP) and mature luminal cells (LC) with distinct gene expression patterns. However, whether these gene expression differences are cell-intrinsic, driven through LP-LC cell interactions or due to stromal-luminal cell interactions in the breasts of healthy women is incompletely understood. Furthermore, prior studies on these cell populations did not take genetic ancestry into consideration despite widely reported genetic ancestry-dependent differences in the normal breast biology and breast cancer susceptibility/outcome. We isolated LP and LC breast epithelial cells from core breast biopsies of healthy women representing European, African American and Latino ancestry (five each). After a brief propagation (five passages) under epithelial reprogramming assay conditions, transcriptomes and DNA methylomes were examined using RNA- and Methylcapture-sequencing. Differences in gene expression (FDR<0.05, fold change >2) in LC cells compared to LP cells were observed, with 1581 and 609 genes up- and down-regulated, respectively. These data were enriched for signaling networks associated with cell cycle control of chromosomal replication, mitotic roles of polo-like kinase (down-regulated genes), granulocyte adhesion and diapedesis and LXR/RXR signaling (up-regulated genes). Despite marked gene expression differences between LC and LP cells, only 14 upregulated genes and four down-regulated genes showed DNA hypo- and hyper-methylation, respectively, in LC cells compared to LP cells, suggesting a minimal role for DNA methylation in differential gene expression between the two cell types. Furthermore, of the 2190 differentially expressed genes, 7% (158 genes) were regulated by estrogen, suggesting that differences in hormone responsiveness contribute to differential gene expression in LP and LC cells. To determine relationships between genes differentially methylated and expressed in LC and LP cells and breast cancer, we used publicly available databases, including TCGA. Interestingly, all genes hypermethylated and expressed at lower levels in LC cells were similarly hypermethylated and expressed at lower levels in breast cancers compared to normal breast, suggesting that these genes are minimally altered in breast cancer. Furthermore, in LC compared to LP cells, of thegenes hypomethylated and expressed at higher levels, only four genes (AIM1L, PRSS27, PTK6 and UNC13D) showed a similar pattern in breast tumors compared to normal breast. Of these, PTK6 is significantly overexpressed in luminal and HER2+ breast cancers compared to basal-like breast cancers, suggesting that PTK6+ cells are the preferred cell-of origin of luminal breast cancers. Taken together, this is the first study to demonstrate that transcriptomic data of normal breast cells from healthy women can be used to further define cell-of-origin of tumors and identify cancer-specific gene expression changes in breast cancer. Citation Format: Fang Fang, Poornima Bhat-Nakshatri, Manjushree Anjanappa, Dough Rusch, Aaron Buechlein, Dave Miller, Kenneth P Nephew, Harikrishna Nakshatri. Intrinsic transcriptomic differences of luminal progenitors and mature luminal cells of the normal breast [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-06-01.

Abstract 4480: Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration

Abstract The Iroquois homeobox 1 (IRX1) belongs to Iroquois homeobox family and plays an important role during embryonic development. Recent studies have suggested that IRX1 acts as a tumor suppressor in gastric cancer and head and neck carcinoma. The zebrafish allows easy genetic modification at a lower cost, thus being a valuable model for cancer biology. For functional annotation we established homozygous knockout mutants of Irx1a and Irx1b and followed to evaluate long-term phenotypes. Homozygous knockout zebrafish for either Irx1a or Irx1b are vital and survived long enough to see long-term phenotypes. However, majority of the homozygotes for both Irx1a /b were severely malformed, could not survive longer than 6 month, and were not fertile. Surprisingly, hyperplasia and tumorigenesis developed in homozygous Irx1a mutants in multiple organs, including bile duct, intestine, kidney, ovary, and testis where irx1 genes are expressed in adults when evaluated by in situ hybridization. Bile duct hyperplasia or tumor was the most common phenotype, occurring in half and two thirds at 6 and 12 months, respectively. Irx1b mutants also developed similar tumorigenesis but showed less prevalent phenotypes than Irx1a mutants, suggesting functional preservation and redundancy. Analysis of differential genes (Wild type vs. Irx1a/b knockout) by cRNA microarray (Agilent Zebrafish v3 GE 4X44 Microarrays) revealed 687 up- and 963 downregulated genes by Irx1a/b knockout. Significant canonical pathways were cyclins and cell cycle regulation, mitotic roles of polo-like kinase, FXR/RXR activation, estrogen-mediated S-phase entry, and cell cycle control of chromosomal replication, suggesting aberrant cell cycle regulation as an important mechanism of tumorigenesis. Real-time RT PCR validated the differential expression of cell cycle-related genes, especially genes involved in G2/M phase progression. Differential genes included CCND1, ARAF, CDKN2C, and FGFR1 among the upregulated genes, and ANAPC7, CCNA2, CCNB1, 2, HAUS1, 3, 6, CDC7, CDC20, CDCA8, FBXO5, 32, 43 and PLK3 among the downregulated genes. Overexpression by transfection decreased cell proliferation in cholangiocarcinoma cells (SNU-1196 and HuCCT1) without altering sensitivity to H2O2-induced apoptosis on annexin V flow cytometry. Western blot revealed decreased expression of CCND1, CCNE, CCNA1, CCNB1 by IRX1 expression. Flow cytometry using propidium iodide and Alexa647-anti-pHH3 showed decreased mitotic fraction by IRX1 expression. On cell cycle synchronization and release using hydroxyuria and nocodazole showed slight delay in G1-S progression and marked delay in G2-M progression and entry into G1. In conclusion, this study identified Irx1 gene as an important tumor-suppressor gene at various organs, and knockout or Irx1 leads to tumorigenesis by altering cell cycle regulation, especially at G2/M phase. Citation Format: Inhye Jung, Dawoon E. Jung, DoHee Kim, Dong Hee Kim, Yong Yoon Chung, Seung Woo Park. Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4480.

Abstract 3569: Comparative RNA-seq analysis reveals dys-regulation of major canonical pathways in ERG-inducible LNCaP cell progression model of prostate cancer

Abstract Prostate Cancer (CaP) is the most common non-cutaneous form of cancer in men and is the second leading cause of cancer mortality in men in the USA. In human CaP, gene fusion between androgen responsive regulatory elements at the 5' untranslated region of TMPRSS2 and ETS-related genes (ERG) is present in at least 50% of the prostate tumors. To identify and investigate the underlying mechanisms of ERG-associated CaP, we developed ERG-inducible LNCaP cell system. In this present study, we investigated the unique cellular transcriptome associated with over-expressed ERG in CaP cells. Our data from comprehensive transcriptome analyses, illustrate a distinct signature that distinguishes ERG-dependent and ERG-independent CaP. The data highlight the significant heterogeneity among the transcript. Out of the 527 differentially expressed genes, 232 genes were up-regulated and 295 genes were down-regulated in response to ERG. Subsequent, in silico analyses indicate that these differentially expressed genes were associated with many pathways and functions. The most significantly differentially expressed genes were associated with cell cycle regulation. The top-ranked biological functions affected by ERG over-expression include Cell Cycle (p < 1.42E-04), Cellular Growth and Proliferation (p < 1.23E-04), Cellular Development (p <1.23E-04), Cell Death and Survival (p < 1.37E-04), and Cellular Assembly and Organization (p < 1.42E-04). Further analyses indicate a strong association with known cancer networks. The top-ranked canonical pathways enriched in ERG-positive compared to ERG-negative LNCaP cells include, Cell Cycle Control of Chromosomal Replication (p=2.69E-16), Role of CHK Proteins in Cell Cycle Checkpoint Control (p=3.16E-11), Cell Cycle: G2/M DNA Damage Checkpoint Regulation (p=1.34E-09), Role of BRCA1 in DNA Damage Response (p=4.05E-08) and Estrogen-mediated S-phase Entry (p =5.51E-08). These findings indicate new insights into the complexity of TMPRESS2-ERG gene fusion, and may help understand mechanistic pathways which promote growth and progression of CaP. Citation Format: Parameet Kumar, Joyeeta Chakraborty, Raghunath Chatterjee, Gauthaman Sukumar, Clifton L. Dalgard, Kevin Babcock, Albert Dobi, Taduru L. Sreenath, Roopa Biswas. Comparative RNA-seq analysis reveals dys-regulation of major canonical pathways in ERG-inducible LNCaP cell progression model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3569. doi:10.1158/1538-7445.AM2017-3569

Kinase gene expression profiling of metastatic tumor tissue to prioritize therapeutic targets in clear cell renal cell carcinoma.

476 Background: Kinase inhibitors are the mainstay of therapy for patients (pts) with metastatic clear cell renal cell carcinoma (ccRCC). Since kinases are actionable and lethal metastatic tumor tissue has not been comprehensively studied, we hypothesized that paired intra-patient kinase gene expression analysis in primary tumor (T), matched normal kidney (N) and metastatic tumor tissue (M) may help identify drivers of metastasis and potential therapeutic targets. Methods: Total mRNA isolated from macrodissected formalin fixed paraffin embedded tissue from T, N, and M from 35 pts (total 105 samples) with ccRCC underwent expression profiling for 519 kinase genes using the nCounter System by Nanostring Technologies. Digital raw counts of mRNA abundance were normalized using internal positive and negative controls as well as 7 housekeeping genes. The mean signals from N, T, and M sites were used to calculate change in gene expression, and a p value by t test <0.05 was considered significant. To identify genes unique to metastasis, kinases with significant expression in the M compared to N or T were selected. Functional analysis was conducted to identify key signaling pathways by using Ingenuity Pathway Analysis. Results: The median age of pts was 56 years and 30 were male. Fifteen pts had metastatic disease at presentation and the remaining developed subsequent metastasis. Clustering analysis for all 105 samples separated normal tissues from tumor or metastasis. The top pathways involved in the kinases overexpressed in M compared to N or T involved Pyridoxal 5'-phosphate Salvage, Salvage Pathways of Pyrimidine Ribonucleotides, NF-kB Signaling, NGF Signaling and Cell Cycle Control of Chromosomal Replication. The top kinases over-expressed in M compared to T include: EPHB2, AURKA, GUCY2C, GSG2, IKBKE, MELK, CSK, CHEK2, CDC7, and MAP3K8. Conclusions: Multiple pathways and kinase genes wereover-expressed in metastatic ccRCC, which suggests a major role for agents targeting metabolic pathways and inhibitors of kinases not currently recognized as therapeutic targets. These data warrant validation and suggest potential drivers of metastasis and actionable therapeutic targets in metastatic ccRCC.

Abstract 859: Regulation of cell cycle and NAD biosynthesis II pathways are associated with differential response to preoperative radiotherapy in locally advanced breast carcinoma

Abstract Human breast carcinomas have heterogeneous pathologies, molecular profiles and different responses to therapy. For non-inflammatory locally-advanced breast cancer patients (LABC), preoperative radiotherapy (pRT) is currently indicated after neoadjuvant chemoherapy (CHT), if there is no response to CHT. However, there are no predictive markers of response to pRT in routine clinical practice. Furthermore, understanding of the molecular mechanisms associated to radiotherapy response could open new avenues for overcoming radio-resistance in these patients. In this retrospective study, we have analyzed genes associated with different response to pRT in a neo-adjuvant setting in LABC patients treated with preoperative RT with a total dose of 45 Gy in 15 fractions every second day to the breast and regional lymph nodes, without previous or concomitant systemic therapy. Radical mastectomy was performed 6 weeks after pRT, and adjuvant systemic therapy was administered as per protocol. Response to pRT was classified as pathological complete response (pCR) if there is no evidence of tumor in the breast after mastectomy, partial response (PR) if regression of tumor was >30%, and no response if regression was <30%. We have analyzed global gene expression profiles by Agilent 8x60K microarray platform in a set of 14 samples of formalin-fixed paraffin-embedded (FFPE) initial LABC biopsies. Unsupervised clustering analysis (Pearson correlation, un-centered metrics) over top 20% most variable genes revealed clear differences in the gene expression profile between radio-resistant and radio-sensitive tumors, despite the small sample size. We have found 165 genes significantly differentially expressed (FDR < 0.10) between responders and non-responders to pRT. To explain the biological processes and signaling pathways of radioresistance, a gene set enrichment using Ingenuity Pathways Analysis was applied. Top scoring molecular functions associated with this gene list included regulation of cellular development, cellular growth and proliferation and cell cycle. Not surprisingly, the most significantly enriched canonical pathways (Fisher's exact p-value<0.05) within the gene list of significantly differentially expressed genes between RT resistant and RT sensitive tumors were cell cycle control of chromosomal replication and pathways related to glucose metabolism and NAD biosynthesis II route. These results set basis for further understanding of molecular response to radiotherapy and highlight genes potentially useful as predictive markers of response to preoperative radiation treatment. Further confirmatory research of potentially RT sensitive/resistant genes and pathways is warranted. Citation Format: Miljana Tanic, Ana Krivokuca, Jasmina Mladenovic, Snezana Susnjar, Sinisa Radulovic, Radmila Jankovic. Regulation of cell cycle and NAD biosynthesis II pathways are associated with differential response to preoperative radiotherapy in locally advanced breast carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 859. doi:10.1158/1538-7445.AM2014-859

Fifty years after the replicon hypothesis: cell-specific master regulators as new players in chromosome replication control.

Numerous free-living bacteria undergo complex differentiation in response to unfavorable environmental conditions or as part of their natural cell cycle. Developmental programs require the de novo expression of several sets of genes responsible for morphological, physiological, and metabolic changes, such as spore/endospore formation, the generation of flagella, and the synthesis of antibiotics. Notably, the frequency of chromosomal replication initiation events must also be adjusted with respect to the developmental stage in order to ensure that each nascent cell receives a single copy of the chromosomal DNA. In this review, we focus on the master transcriptional factors, Spo0A, CtrA, and AdpA, which coordinate developmental program and which were recently demonstrated to control chromosome replication. We summarize the current state of knowledge on the role of these developmental regulators in synchronizing the replication with cell differentiation in Bacillus subtilis, Caulobacter crescentus, and Streptomyces coelicolor, respectively.

Abstract 3283: Conditional Low-molecular-weight (LMW) cyclin E-induced mammary tumorigenesis

Abstract Cyclin E overexpression and generation of hyperactive LMW cyclin E is oncogenic and has been implicated in a variety of human cancers. We have developed a doxycycline-inducible LMW cyclin E transgenic mouse model to determine the dependence of established mammary adenocarcinomas on continued expression of LMW cyclin E. Mice harboring this TetO-LMW-E transgene, referred to as TLMW, were mated to MMTV-rtTA transgenic mice (MTB) to yield bitransgenic MTB/TLMW mice. Morphological examination of hematoxylin and eosin-stained sections showed that the mammary glands of induced MTB/TLMW mice following four days of doxycycline induction were hyperplastic compared with uninduced MTB/ TLMW animals. Consistent with this, immunohistochemistry using antibodies specific for BrdU revealed a 2-fold increase in BrdU incorporation in the mammary epithelium of induced bitransgenic females compared with uninduced mice (17.1+/−5.3% versus 30.9+/−4.9%, P=0.03). This data indicate that LMW cyclin E overexpression in the mammary epithelium increased proliferation. To determine whether sustained induction of transgene expression would result in mammary tumorigenesis in MTB/TLMW mice, a cohort of 27 bitransgenic females was treated with doxycycline beginning at 6 weeks of age. 41% of these mice developed tumors within 2 years of starting doxycycline with kinetics (T1/2= 20 months) resembling that described for MMTV-LMW cyclin E mice. Similarly, the predominant type of tumor observed for the inducible LMW forms was glandular adenocarcinomas with adenosquamous differentiation. Lung metastases are known to arise in a fraction of MMTV-LMW cyclin E transgenic mice harboring primary mammary tumors. Accordingly, we sacrificed a subset of mammary tumor bearing MTB/TLMW mice and examined organs at necropsy for evidence of metastatic spread. 4 of 7 mice examined had lung metastasizes (57%). Control mice without dox never developed mammary tumors. To shed further light on the molecular mechanisms of mammary tumorigenesis caused by LMW-E expression, we conducted DNA microarray analyses. Using the R package limma, 641 probes out of 30,854 probes were identified as differentially regulated by at least 2-fold by LMW-E in the tumors when compared to the contralateral mammary glands. Gene Ontology analysis identified genes on this list that were involved in a variety of processes, including cell cycle control of chromosome replication, and DNA methylation and transcriptional repression signaling. Based on histology and gene expression profile of the resultant mammary tumors, we propose that the MTB/TLMW is a murine model of breast cancer resembling the human invasive basal/triple negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3283. doi:1538-7445.AM2012-3283

Chromosomal Macrodomains and Associated Proteins: Implications for DNA Organization and Replication in Gram Negative Bacteria

The Escherichia coli chromosome is organized into four macrodomains, the function and organisation of which are poorly understood. In this review we focus on the MatP, SeqA, and SlmA proteins that have recently been identified as the first examples of factors with macrodomain-specific DNA-binding properties. In particular, we review the evidence that these factors contribute towards the control of chromosome replication and segregation by specifically targeting subregions of the genome and contributing towards their unique properties. Genome sequence analysis of multiple related bacteria, including pathogenic species, reveals that macrodomain-specific distribution of SeqA, SlmA, and MatP is conserved, suggesting common principles of chromosome organisation in these organisms. This discovery of proteins with macrodomain-specific binding properties hints that there are other proteins with similar specificity yet to be unveiled. We discuss the roles of the proteins identified to date as well as strategies that may be employed to discover new factors.

Epigenética del cáncer

The pathogenesis of cancer includes both genetic and epigenetic mechanisms. The term <<epigenetic>> refers to any alteration of gene expression, potentially hereditary, which is not accompanied by a modification in the DNA sequence. The effects of epigenetic changes include alteration of DNA transcription, aberrant activation of specific genes, predisposition of genetic instability through alteration of the control of chromosome replication and silencing of the genes implicated in cancer initiation and progression. Among the various epigenetic alterations that lead to altered gene expression, methylation is the main epigenomic mechanism implicated in cancer, whether through a phenomenon of overall hypomethylation or hypermethylation localized in the promoters of specific genes.

DivL Performs Critical Cell Cycle Functions inCaulobacter crescentusIndependent of Kinase Activity

The Caulobacter cell cycle is regulated by a network of two-component signal transduction proteins. Phosphorylation and stability of the master transcriptional regulator CtrA are controlled by the CckA-ChpT phosphorelay, and CckA activity is modulated by another response regulator, DivK. In a screen to identify suppressors of the cold-sensitive divK341 mutant, we found point mutations in the essential gene divL. DivL is similar to histidine kinases but has a tyrosine instead of a histidine at the conserved phosphorylation site (Y550). Surprisingly, we found that the ATPase domain of DivL is not essential for Caulobacter viability. We show that DivL selectively affects CtrA phosphorylation but not CtrA proteolysis, indicating that DivL acts in a pathway independent of the CckA-ChpT phosphorelay. divL can be deleted in a strain overproducing the phosphomimetic protein CtrAD51E, but unlike DeltactrA cells expressing CtrAD51E, this strain is profoundly impaired in the control of chromosome replication and cell division. Thus, DivL performs a second function in addition to promoting CtrA phosphorylation. DivL is required for bipolar DivK localization and positively regulates DivK phosphorylation. Our results show that DivL controls two key cell cycle regulators, CtrA and DivK, and that phosphoryl transfer is not DivL's essential cellular activity.

Cluster of DnaA boxes involved in regulation of Streptomyces chromosome replication: from in silico to in vivo studies.

In Streptomyces coelicolor, replication is initiated by the DnaA protein in the centrally located oriC region and proceeds bidirectionally until the replication forks reach the ends of the linear chromosome. We identified three clusters of DnaA boxes (H69, H24, and D78) which are in a relatively short segment of the chromosome centered on the oriC region. Of the clusters analyzed, D78 exhibited the highest affinity for the DnaA protein; the affinity of DnaA for the D78 cluster was about eightfold higher than the affinity for oriC. The high-affinity DnaA boxes appear to be involved in the control of chromosome replication. Deletion of D78 resulted in more frequent chromosome replication (an elevated ratio of origins to chromosome ends was observed) and activated aerial mycelium formation, leading to earlier colony maturation. In contrast, extra copies of D78 (delivered on a plasmid) caused slow colony growth, presumably because of a reduction in the frequency of initiation of chromosome replication. This suggests that the number of high-affinity DnaA boxes is relatively constant in hyphal compartments and that deletion of D78 therefore permits an increased copy number of either the chromosomal origin region or a plasmid harboring the D78 cluster. This system conceivably influences the timing of decisions to initiate aerial mycelial formation and sporulation.

OriC Region and Replication Termination Site, dif , of the Xanthomonas campestris pv. campestris 17 Chromosome

A 13-kb DNA fragment containing oriC and the flanking genes thdF, orf900, yidC, rnpA, rpmH, oriC, dnaA, dnaN, recF, and gyrB was cloned from the gram-negative plant pathogen Xanthomonas campestris pv. campestris 17. These genes are conserved in order with other eubacterial oriC genes and code for proteins that share high degrees of identity with their homologues, except for orf900, which has a homologue only in Xylella fastidiosa. The dnaA/dnaN intergenic region (273 bp) identified to be the minimal oriC region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical oriCs contain four or more DnaA boxes located upstream of the dnaA gene. The X. campestris pv. campestris 17 attB site required for site-specific integration of cloned fragments from filamentous phage phiLf replicative form DNA was identified to be a dif site on the basis of similarities in nucleotide sequence and function with the Escherichia coli dif site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The oriC and dif sites were located at 12:00 and 6:00, respectively, on the circular X. campestris pv. campestris 17 chromosome map, similar to the locations found for E. coli sites. Computer searches revealed the presence of both the dif site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the dif sites are located far away from the 6:00 point instead of being placed opposite the cognate oriC. The differences in the relative position suggest that mechanisms different from that of E. coli may participate in the control of chromosome replication.

Host cell variations resulting from F plasmid-controlled replication of the Escherichia coli chromosome.

Cell size and DNA concentration were measured in Escherichia coli K-12 ET64. This strain carries a dnaA (Ts) mutation that has been suppressed by the insertion of the F plasmid into the chromosome. ET64 can grow in a balanced steady state of exponential growth at the restrictive temperature for its dnaA allele (39 degrees C), in which chromosome replication is controlled by the F plasmid, and at the permissive temperature (30 degrees C), in which chromosome replication is controlled by dnaA-oriC. When cells grown at the indicated temperatures were compared, it was observed that at 39 degrees C, the cell mass increased and the amount of cellular DNA decreased slightly; therefore, the DNA concentration was strongly reduced. These changes can neither be explained by the reduction of the generation time (which is only 10-15%) nor from observed changes in the replication time and in the time between DNA synthesis termination and cell division. Variations were mainly due to the increase in cell mass per origin of replication, at initiation, in cells grown at 39 degrees C. Control of chromosome replication by the F plasmid appears to be the reason for the increase in the initiation mass. Other possible causes, such as the modification of growth temperature, the generation time, or both, were discarded. These observations suggest that at one growth rate, the F plasmid replicates at a particular cell mass to F particle number ratio, and that this ratio is higher than the cell mass to oriC ratio at the initiation of chromosome replication. This fact might be significant to coordinate the replication of two different replicons in the same cell.

Control of chromosome replication in bacteria.

The exciting progress that has recently been made in unravelling the replication control mechanisms that determine the copy numbers of several plasmids has been reviewed and updated by several contributors to this volume. In contrast, the control of chromosome replication in bacteria remains largely a closed book.KeywordsChromosome ReplicationdnaA StrainExciting ProgressInitiation ControloriC PlasmidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Accelerated chromatin replication in embryonic cells

Early animal embryos replicate their chromosomes and divide faster than E.coli replicates its much smaller chromosome. The control of chromosome replication in Xenopus embryos has been investigated by studying the replication of microinjected DNA. Replication of injected DNA is regulated by a cytoplasmic cell cycle clock. The pattern of regulation can be changed by the tumour promoter, TPA.

Relation between growth and replication in bacteria

The control of chromosome replication in bacteria depends on the cell growth; a round of replication is initiated when the cell mass (or protein) per replication origin has reached a certain critical level. Relative values of this “initiation mass” have previously been estimated from the evaluation of experiments involving age fractionation of radioactively labelled bacterial cultures. Here a theoretical analysis is presented which permits a simple and accurate method to determine this initiation mass from a few parameters easily observable with exponential cultures, without the need for age fractionation.

Control of chromosome replication in thymine-requiring strains of Bacillus subtilis 168.

Study of the replication pattern of a number of B. subtilis 168 strains under controlled physiological conditions revealed great interstrain variation in control of replication. Replication patterns were calculated from ratios of purA16/leu-8 and purA16/metB5 transformation frequency. The thymine-independent strains are under strict regulation with an average of one replication position per chromosome during log phase. After starvation for required amino acids or sporulation, the chromosome is in a completed state with no replication forks (class I). In contrast, several thymine-requiring strains (class III) have an average of three to four replication positions per chromosome during log phase (multiforked replication) of which one to two remain uncompleted after amino acid starvation or sporulation. The other thymine-requiring strains studied are intermediate (class II) in that they have an average of two replication positions per chromosome during log phase and one after amino acid starvation or sporulation. Pulse chase experiments indicate that the deoxyribonucleic acid which is close to the chromosomal origin on each branch of the multiforked chromosome is bound to a rapidly sedimenting cellular fraction, presumably membrane.

Chromosome replication in Escherichia coli: IV. Control of chromosome replication and cell division by an integrated episome

A temperature-sensitive mutation (T46) affecting the initiation of DNA replication in Escherichia coli is not complemented by an F factor. If, however, temperatureresistant revertants are isolated from a T46 s F + strain most of them are Hfr, with F integrated at various sites. These Hfr temperature resistant cells still contain the T46 s mutation. They are sensitive to acridine orange and ethidium bromide at 42 °C, but not at 30 °C, suggesting that, at the restrictive temperature, they are under the control of the F replicon. Similar results were obtained in a partial diploid strain carrying the F′ lac factor. In a large number of the Hfr temperature resistant strains formed, F′ lac had integrated in the lac region of the chromosome, but Hfr temperature resistant strains were also produced by integration of F′ lac at other sites. “Integrative suppression” by F′ lac was also observed for another temperaturesensitive mutation affecting the initiation of DNA replication (T83). It requires the function specified by the recA gene. Integrative suppression does not take place for mutations which affect the continuation of DNA replication rather than its initiation. F integration alone is not sufficient for integrative suppression and another step is needed. The nature of that step remains unknown. We conclude that, in the Hfr temperature resistant strains, chromosome replication and cell division are under F control. It seems that in these cells the chromosome has become part of the F replicon.

Genetic effects of acridines on Pseudomonas aeruginosa.

Mutants ofPseudomonas aeruginosamale strains with altered mating properties have been obtained through treatment with nitrogen half mustards. The response varied with the type of male strain. A mutant of the infectious male PTO 13 was obtained which acts as a female strain and has apparently lost the FP sex factor. A mutant of the non-infectious strain 2 male mates not only with strain 1 females, but shows a thousand-fold increase in its ability to mate with strain 2 males, when it can act as either donor or recipient in conjugation. Derivatives of strain 1 males were obtained which had reduced recombinant forming ability.While acriflavine is ineffective in producing such mutants, and has not been shown to cure male strains of their sex factor inP. aeruginosa, it is very effective in inhibiting infectious transfer of FP from FP+to FP−strains. Furthermore, it markedly inhibits recombinant formation in 1 FP−× 1 FP+crosses, and this latter effect is thought to be due to the inhibition of chromosome transfer by the male parent. In view of the almost complete lack of effect of AF on the normal growth ofP. aeruginosait is likely that the control of chromosome replication during vegetative cell division is different from the occurring during conjugation and that the FP factor is involved in this control during conjugation.