Pattern Of Chromosome Replication Research Articles

Fifty-Five Years of Research on B, C and D in Escherichia coli.

The basic properties of the Escherichia coli duplication process can be defined by two time periods: C, the time for a round of chromosome replication, and D, the time between the end of a round of replication and cell division. Given the durations of these periods, the pattern of chromosome replication during the cell cycle can be determined for cells growing with any doubling time. In the 55 years since these parameters were identified, there have been numerous investigations into their durations and into the elements that determine their initiations. In this review, I discuss the history of our involvement in these studies from the very beginning, some of what has been learned over the years by measuring the durations of C and D, and what might be learned with additional investigations.

Genome-wide identification, characterization, and expression pattern of the late embryogenesis abundant (LEA) gene family in Juglans regia and its wild relatives J. mandshurica

BackgroundLate Embryogenesis Abundant (LEA) proteins are a class of proteins associated with plant stress resistance. Two Juglans species, Juglans regia and J. mandshurica, are both diploid (2n = 32), monoecious perennial economic tree species with high edible, pharmaceutical, and timber value. The identification, characterization, and expression patterns of LEA proteins in J. regia and its wild relative, J. mandshurica, would not only provide the genetic basis of this gene family, but it would also supply clues for further studies of the evolution and regulating mechanisms of LEA proteins in other tree species.ResultsIn this study, we identified 25 and 20 members of the LEA gene family in Juglans regia and its wild relative, Juglans mandshurica, respectively. The results of phylogenetic analysis showed that the LEA members were divided into eight main subgroups. Predictions of their physicochemical properties showed the variable characteristics of LEA proteins, and the subcellular localization analysis indicated that most LEA proteins are localized in the nucleus. Chromosomal localization analysis and gene replication pattern prediction indicated that WGD is the predominant duplication mode of LEA genes. The results of the comparative analysis indicated a high level of collinearity between the two Juglans species. Analysis of cis-acting elements indicated that LEA genes had a relatively wide range of responses to abiotic stresses and phytohormonal processes, particularly in two phytohormones, methyl jasmonate and abscisic acid. Transcriptome profiling and qRT-PCR experiments showed that JrLEAs are commonly expressed in leaves, green husks, and male and female flowers, and most JmLEAs are more highly expressed in male flowers. We also hypothesized that JrLEAs are involved in the process of anthracnose resistance. Anthracnose-resistant varieties of JrLEAs presented relatively high expression levels at later stages.ConclusionIn this study, we provide a theoretical basis for the functional study of LEA genes in J. regia and J. mandshurica. Analysis of cis-acting elements and gene expression indicated that JrLEAs and JmLEAs play important roles in resistance to biotic stresses in these species.

Chromosomal Mcm2-7 distribution and the genome replication program in species from yeast to humans.

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.

A ten-year search for synchronous cells: obstacles, solutions, and practical applications.

My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end. In this essay, I recount my personal journey in this obsessive experimental pursuit. That narrative is followed by a description of a simplified version of the “baby machine,” a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells. Subsequent studies with this methodology led to an understanding of the basic properties of the relationship between chromosome replication and cell division. Accordingly, I end this reminiscence with a simple, fool-proof graphical strategy for deducing the pattern of chromosome replication during the division cycle of cells growing at any rate.

Maintaining replication origins in the face of genomic change

Origins of replication present a paradox to evolutionary biologists. As a collection, they are absolutely essential genomic features, but individually are highly redundant and nonessential. It is therefore difficult to predict to what extent and in what regard origins are conserved over evolutionary time. Here, through a comparative genomic analysis of replication origins and chromosomal replication patterns in the budding yeasts Saccharomyces cerevisiae and Lachancea waltii, we assess to what extent replication origins survived genomic change produced from 150 million years of evolution. We find that L. waltii origins exhibit a core consensus sequence and nucleosome occupancy pattern highly similar to those of S. cerevisiae origins. We further observe that the overall progression of chromosomal replication is similar between L. waltii and S. cerevisiae. Nevertheless, few origins show evidence of being conserved in location between the two species. Among the conserved origins are those surrounding centromeres and adjacent to histone genes, suggesting that proximity to an origin may be important for their regulation. We conclude that, over evolutionary time, origins maintain sequence, structure, and regulation, but are continually being created and destroyed, with the result that their locations are generally not conserved.

No Evidence for “Break-Induced Replication” in a Higher Plant – But Break-Induced Conversion May Occur

“Break-induced replication” (BIR) is considered as one way to repair DNA double-strand breaks (DSBs). BIR is defined as replication of the proximal break-ends up to the end of the broken chromosome using an undamaged (homologous) double-stranded template and mimicking a non-reciprocal translocation. This phenomenon was detected by genetic experiments in yeast. BIR is assumed to occur also in mammals, but experimental evidence is not yet at hand. We have studied chromosomes of the field bean, Vicia faba L., with respect to the occurrence of BIR after DSB induction during S and G2 phase. Simultaneous incorporation of the base analog ethynyldeoxyuridine (EdU) revealed no chromosomal replication pattern indicative of BIR. Thus, if occurring at all, BIR does not play a major role in DSB repair in higher plants with large chromosome arms. However, the frequency of interstitial asymmetric EdU incorporation within heterochromatic regions, visible on metaphase chromosomes, increased after chromosome breakage during S and G2 phase. Such asymmetric labeling could be interpreted as conservative replication up to the next replicon, circumventing a DSB, and yielding an interstitial conversion-like event.

CHROMOSOME DNA SYNTHESIS IN THE CELLS OF A HUMAN TRIPLOID/DIPLOID MOSAIC

HereditasVolume 55, Issue 1 p. 55-67 Open Access CHROMOSOME DNA SYNTHESIS IN THE CELLS OF A HUMAN TRIPLOID/DIPLOID MOSAIC LEONARD ATKINS, LEONARD ATKINS INSTITUTE FOR MEDICAL GENETICS, UPPSALA, SWEDEN Massachusetts General Hospital, Boston, Massachusetts, 02114, U.S.A.Search for more papers by this authorJ. A. BÖÖK, J. A. BÖÖK INSTITUTE FOR MEDICAL GENETICS, UPPSALA, SWEDENSearch for more papers by this authorBERTA SANTESSON, BERTA SANTESSON INSTITUTE FOR MEDICAL GENETICS, UPPSALA, SWEDENSearch for more papers by this author LEONARD ATKINS, LEONARD ATKINS INSTITUTE FOR MEDICAL GENETICS, UPPSALA, SWEDEN Massachusetts General Hospital, Boston, Massachusetts, 02114, U.S.A.Search for more papers by this authorJ. A. BÖÖK, J. A. BÖÖK INSTITUTE FOR MEDICAL GENETICS, UPPSALA, SWEDENSearch for more papers by this authorBERTA SANTESSON, BERTA SANTESSON INSTITUTE FOR MEDICAL GENETICS, UPPSALA, SWEDENSearch for more papers by this author First published: August 1966 https://doi.org/10.1111/j.1601-5223.1966.tb02035.xCitations: 2 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Literature cited Atkins, L., Gustavson, K.-H. and Hansson, O. 1963. A case of XXXXY sex chromosome anomaly with autoradiographic studies. Cytogenetics 2: 208– 232. Atkins, L. and Santesson, Berta. Chromosome DNA synthesis in cultured normal human female skin cells. Hereditas 55: 39– 46. Böök, J. A., Atkins, L. and Santesson, Berta 1963. Some new data on autosomal aberrations in man. Path. Biol. 11: 1159– 1162. Böök, J. A., Masterson, J. B. and Santesson, Berta 1962. Malformation syndrome associated with triploidy—further chromosome studies of the patient and his family. Acta Genet. 12: 193– 201. Böök, J. A. and Santesson, Berta 1960. Malformation syndrome in man associated with triploidy (69 chromosomes). Lancet i: 858– 859. Böök, J. A. and Santesson, Berta 1961. Nuclear sex in triploid XXY human cells. Lancet ii: 318. Böök, J. A., Santesson, Berta and Zetterqvist, P. 1961. Association between congenital heart malformation and chromosomal variations. Acta Paediat. Sthlm. 50: 217– 227. Carr, D. H. 1963. Chromosome studies in abortuses and stillborn infants. Lancet ii: 603– 606. Ferrier, P., Ferrier, S., Stalder G., BÜhxer, E., Bamatter, F. and Klein, D. 1964. Congenital asymmetry associated with diploid-triploid mosaicism and large satellites. Lancet i: 80– 82. German, J. 1963. The pattern of DNA synthesis in the chromosomes of human blood cells. J. Cell. Biol. 20: 37– 55. German, J., Lejeune, J., Macintyre, M. N. and Degrouchy, J. 1964. Chromosomal autoradiography in the Cri du Chat syndrome. Cytogenetics 3: 347– 352. Miller, O. J., Mukherjee, B. B., Breg, W. R. and Gamble, A. van N. 1963. Non-random distribution of chromosomes in metaphase figures from cultured huma leucocytes. I. The peripheral location of the Y chromosome. Cytogenetics 2: 1– 14. Mittwoch, U. and Delhanty, J. D. A. 1961. Nuclear sex in triploid XXY human cells. Lancet ii: 552. Moorhead, P. S. and Defendi, V. 1963. Asynchrony of DNA synthesis in chromosomes of human diploid cells. J. Cell. Biol. 16: 202– 209. Saksela, E. and Moorhead, P. S. 1962. Enhancement of secondary constrictions and the heterochromatic X in human cells. Cytogenetics 1: 225– 244. Schmid, W. 1963. DNA replication patterns of human chromosomes. Cytogenetics 2: 175– 193. Yunis, J. J., Hook, E. B. and Mayer, M. 1964. Deoxyribonucleic-acid replication pattern of trisomy D1 Lancet ii: 935– 936. Citing Literature Volume55, Issue1August 1966Pages 55-67 ReferencesRelatedInformation

DNA replication patterns of canine chromosomes in vivo and in vitro.

DNA replication patterns of canine chromosomes in vivo and in vitro.

Identification of heterochromatic regions in the chromosomes of rye

Localisation of the heterochromatic regions in the chromosomes of rye, Secale cereale L., has been achieved by (a) C-banding and (b) fluorescence banding with a bis-benzimidazole derivative. It is possible to characterise each pair of the rye chromosomes. The absence of heterochromatin at the centromeric regions and its presence in the terminal ends of the chromosomes, serve as markers and facilitate easy identification of rye chromosomes from those of wheat, in Triticale genome. Though the modern staining techniques have been effectively utilized in the identification of individual chromosomes in several mammals, their use in plant cytology has been of limited success. Quinacrine banding (Q-banding) of the heterochromatic regions of the chromosomes has been reported in some plants by CASPERSSON et al. 1969, VOSA 1970 and HORN and WALDEN 1971. In addition, NATARAJAN and NATARAJAN (1972) have shown in Rhoeo discolor that the Q-positive regions are also C-positive, i.e., these chromosomal regions take up heavily Giemsa stain, following denaturation and reassociation of cytological preparations. Secale cereale L., is known to possess well defined heterochromatic regions, as revealed by the pattern of chromosome replication (LIMA DE FARIA 1959). This species has been combined with the wheat genomes to produce Triticale and many addition and substitution lines have been produced involving wheat genomes to which one pair of rye chromosomes is separately added or substituted to the normal complement (RILEY and CHAPMAN 1958; BEILIG and DRISCOLL 1970). Rye has also been used to study the role of heterochromatin in the formation of chromosome aberrations, following treatments with radiations and chemicals (NATARAJAN and AHNSTROM 1970, 1972). Identification of individual rye chromosomes will be of practical and theoretical value. The results of our attempts to characterise the rye chromosomes are presented and discussed in this communication.

Dynamics ofEscherichia coliChromosome Segregation during Multifork Replication

Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive cohesion of sister DNA regions was seen at any growth rate. We conclude that segregation is driven by the progression of the replication forks.

Jake, Leon O. Jacobson, M.D.: The Life and Work of a Distinguished Medical Scientist (review)

Reviewed by: Jake, Leon Jacobson, M.D.: The Life and Work of a Distinguished Medical Scientist Janet D. Rowley Jake, Leon Jacobson, M.D.: The Life and Work of a Distinguished Medical Scientist. By Eugene Goldwasser. New York: Science History Publications, 2006. Pp. 96. $14.95. Leon Jacobson—"Jake"—was a special person whose origins in rural North Dakota had a critical influence on how he later behaved in the world of high-powered science. This biography, by Jake's long-time associate and colleague Eugene Goldwasser, traces Jake's evolution from a farm boy to a member of the National Academy of Sciences and a founding member of the Institute of Medicine. Jake was born in 1911 in Sims, North Dakota, a largely agricultural region, with small farms as well as cattle and sheep ranches. His parents were Norwegian, and despite their need for help on the farm from their seven children, the four youngest, including Jake, graduated from high school. Jake was fortunate to have had teachers in grammar school and high school who recognized his special qualities. They tutored and encouraged him to take the next step, and he entered North Dakota Agriculture College in Fargo in 1928. Despite the Depression, failed banks, and the loss of his savings, Jake graduated from college and entered medical school at the University of Chicago in 1935. There was one transforming event during this period: Jake taught eight grades of elementary school in a two-room school near Sims. He described it as, in many ways, the most rewarding three years of his life. [End Page 629] Jake's career after medical school was changed by a series of improbable events. As a second-year resident at the University of Chicago, he became responsible for monitoring the blood counts of physicists working on the Manhattan Project at the university. He was then given responsibility as a physician for all of the staff in the Metallurgical Laboratory, which was the code name given to that part of the Manhattan Project at the university. He did not know exactly what they were doing and thus could not assess their potential risk of any illness. In response to his persistent questions about the activities of the staff, he was finally informed that they were working on the atomic bomb. He was stunned by this revelation but realized that he could not withdraw. As the number of staff increased so did his responsibilities. His own staff also increased, and as a result Jake became more removed from the daily clinical responsibilities and could begin research on the effect of radiation on blood cell formation. Through his contacts with the Manhattan Project and subsequently with friends in various government-sponsored labs around the country, Jake and the then Dean, Lowell T. Coggeshall, proposed to build a cancer research hospital for treating patients with radioisotopes produced by the Atomic Energy Commission (AEC). After some very negative reactions from the AEC, the proposal was approved by Congress. The Argonne National Laboratories, operated by the University of Chicago about 30 miles from the campus, did not want a hospital within its confines, so the Argonne Cancer Research Hospital (ACRH) was incorporated into the university hospital systems. ACRH had an unusual structure for American biomedical operations. It was funded by money from the AEC; as Director, Jake had relative freedom to manage it and to allocate money as he saw fit. ACRH was a paradise because grant applications were simple. There were annual reports and regular program reviews, so Jake could not get too far offbase, but at the same time, he had a remarkable degree of freedom about who to hire and the direction of the research. I consider myself an extreme example. I knew Jake when I was a medical student. After graduating from medical school in 1948, I interned and then worked in well baby clinics and a clinic for retarded children. This latter involvement with retarded children, especially those with Down's syndrome, led me to study chromosomes during my husband's sabbatical, spent in Oxford, 1961–62. On returning to Chicago with my Oxford research project on the pattern of human chromosome replication less...

Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites: what's the Dif?

In this issue of Molecular Microbiology, Hendrickson and Lawrence analyse the sequence of bacterial genomes to map the historical traffic pattern of chromosome replication. Their surprising conclusion is that most forks terminate at the dif site rather than at the Tus/Ter sites where most investigators have concluded termination occurs most frequently. What make this analysis novel are the methods and the revisionist hypotheses for how and why forks might stop at dif.

Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes.

Telomeres are defining structural elements of all linear chromosomes, yet information concerning the timing of their replication in higher eukaryotes is surprisingly limited. We developed an approach that allowed a study of telomere replication patterns of specific mammalian chromosomes. In the Indian muntjac (Muntiacus muntjac), replication timing between respective telomeres of homologous chromosomes was highly coordinated, but no such synchrony was evident for p- and q-arm telomeres of the same chromosome. This finding contrasts with the coordinated timing of both ends of each chromosome in yeast. Also in contrast to yeast, where replication of all telomeres is confined to late S phase, we found specific telomeres in Indian muntjac chromosomes that replicated early in S and other telomeres that replicated later. Finally, replication timing of some but not all telomeres was influenced by telomere length. Knowledge of telomere replication timing represents a first step toward understanding the relationship between telomere replication and telomerase action. The approach, which we call replicative detargeting fluorescence in situ hybridization, is widely applicable to different species and genetic loci.

Replication Patterns of Sex Chromosomes in Melandrium Album Female Cells

Autoradiographic analysis of chromosome replication patterns in heterogametic male and homogametic female hairy-root cell lines of a dioecious plant, Melandrium album, has shown an early replication of the only X and a conspicuous difference between the two X chromosomes, respectively. This divergence could be significantly reduced by a 5-azacytidine treatment, thus confirming our recently published results on the different degree of DNA methylation of the X chromosomes in female cells. The early replicating and aberration prone X chromosome seems to be less condensed, too. [3H]thymidine labelling patterns as well as [3H]uridine induced chromosome aberrations do not indicate any differences between the long and short arms of the X chromosomes. The presented data support the hypothesis that one of the two X chromosomes in M. album female cells could be inactivated and hypermethylated as described in mammals.

Werner Schmid 1930–2002

The Swiss Society of Medical Genetics (SGMG) was founded in 1978 on the initiative of Prof. Werner Schmid, Zurich and Prof. Ed. Juillard, Lausanne. Werner Schmid was the first president and served the Society in many functions until receiving emeritus status in 1995. He was also the long-standing editor of the information bulletin published by the SGMG, with hardly an issue appearing without one or more contributions penned by him. Representing the professional interests of geneticists and transmitting genetic information to the medical profession was his lifelong concern. For example, the inclusion of genetic tests in the Federal Analysis List was due entirely to his initiative. Werner Schmid died on the 3rd of January at the age of 72, after a short illness. The Swiss Society of Medical Genetics owes him a great debt of thanks. In 1967, Werner Schmid became lecturer in medical genetics at Zurich University (postdoctoral thesis: “Heterochromatin in Mammals”), in 1969 assistant professor, 1974 associate professor and in 1978 professor and director of the newly founded Institute of Medical Genetics. In 1981, the Institute separated from the Children's Hospital. In spring 1995 he became emeritus professor. In research, Werner Schmid especially made a name for himself due to the development of new methods. His article published in 1963, “DNA replication patterns of human chromosomes,” caused a worldwide sensation. Observation of the replication process using autoradiography allowed the differentiation of apparently identical human chromosomes at a time when banding techniques were still unknown. His article “Sex chromatin in hair roots” (1967) also began an era of biochemical testing using this easily sampled material. Werner Schmid's “The micronucleus test” (1975), a method also used by the pharmaceutical industry for in vitro and in vivo mutagenicity testing during preclinical and clinical development of new drugs, was a “Citation Classic” in “Current Contents” (1990). The publication “A technique for in situ karyotyping of primary amniotic fluid cell cultures” (1975) had an equally worldwide circulation in prenatal diagnosis. The last of his more than 160 scientific publications to be mentioned here is the so-called “Winterthur Study” (1982), which received national acclaim from teachers and educationalists. In the late 1970s and early 1980s, Werner Schmid and a number of graduate students and assistants carried out a study on “Genetic, medical and psychosocial factors in an age-class of 11 year old Winterthur school children.” A third of the age group did not reach the requirements of the school and were given special teaching, indicating exaggerated selection. Children with difficult home and family lives were those who did not pass selection. Constitutional advantages measured by the professional success of parents and close relatives were hardly noticeable during the first four school years. Since the 1970s, Werner Schmid's aim was to make the new achievements of medical genetics and genetic counseling available as a service to the public. Through countless newspaper and magazine articles, presentations, and podium discussions, he tried to explain the complex field of medical genetics and to open it up to the general public. He was one of the pioneers of prenatal diagnosis in Europe, with his institute being one of the few that, since 1971, has systematically followed up and published data on the progress of pregnancies, births, and general health of children after invasive prenatal diagnosis. In 1985, as his Institute reached capacity and the government refused funding for further staffing, Werner Schmid and a group of doctors founded Genetica AG in Zurich. He gave his support to this laboratory to promote his lifelong aim that everyone should have access to medical genetic services. Werner Schmid dedicated a great deal of time and energy to building up and improving his Institute in a time when state funding was becoming tighter, and made intensive efforts to ensure the constant further education of his co-workers. Countless guest workers from all over the world were accepted into his Institute and were introduced to the secrets of the latest methods. In genetic counseling sessions, Schmid was a patient listener and advisor, even in situations that reached beyond the narrow boundaries of genetics. After being given emeritus status in Spring 1995, Werner Schmid traveled a lot and occupied himself with his interests in world and art history. He was able to read many books that he had previously set aside and enjoyed caring for his beautiful garden. Sadly, he was not granted very much time for this.

Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii.

We analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus(Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea.

Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants.

The accurate duplication and packaging of the genome is an absolute prerequisite to the segregation of chromosomes in mitosis. To understand the process of cell-cycle chromosome dynamics further, we have performed the first detailed characterization of a mutation affecting mitotic chromosome condensation in a metazoan. Our combined genetic and cytological approaches in Drosophila complement and extend existing work employing yeast genetics and Xenopus in vitro extract systems to characterize higher-order chromosome structure and function. Two alleles of the ORC2 gene were found to cause death late in larval development, with defects in cell-cycle progression (delays in S-phase entry and metaphase exit) and chromosome condensation in mitosis. During S-phase progression in wild-type cells, euchromatin replicates early and heterochromatin replicates late. Both alleles disrupted the normal pattern of chromosomal replication, with some euchromatic regions replicating even later than heterochromatin. Mitotic chromosomes were irregularly condensed, with the abnormally late replicating regions of euchromatin exhibiting the greatest problems in mitotic condensation. The results not only reveal novel functions for ORC2 in chromosome architecture in metazoans, they also suggest that the correct timing of DNA replication may be essential for the assembly of chromatin that is fully competent to undergo mitotic condensation.

Functional and Structural Chromosome Analyses in Autotetraploid Silene latifolia

Recent immunocytological and molecular data show that heterochromatic nuclear regions, both constitutive and facultative, are modified differently (cytosine hypermethylation and histone hypoacetylation) and late replicating, when compared to euchromatin. Intrusive and/or additive (supernumerary) DNA sequences are often functionally silenced; this is accompanied by their heterochromatinization. In this work we present a number of karyological studies on autotetraploid female cells of Silene latifolia (syn. Melandrium album). Immunofluorescence analyses do not indicate any global differences in DNA methylation, histone H4 acetylation, and chromosome replication patterns which could arise as a consequence of the duplication of the whole chromosome set of the original diploid genome. Similarly, the number of silver-positive nucleoli roughly correlates to the ploidy level. Early replication and H4 hyperacetylation have been detected at all subterminal chromosome regions. This, together with cDNA in situ hybridization patterns, indicates the localization of gene-rich regions. DNA methylation and chromosome replication patterns, but not histone H4 acetylation, show differences among the four X chromosomes present: one to three X chromosomes were observed as hypermethylated and/or late replicating. Taken together, the data demonstrate that there is no overall silencing of the additional two sets of autosomes in the tetraploid cells, but the X chromosomes could be subject to an irregular dosage compensation.

Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli.

The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry. Chromosome replication patterns in these strains were followed by marker frequency analyses. In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild-type parent. The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication. The site for conversion of uni- to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC. Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi- or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern. These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects. Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality. However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.

Sequence of DNA replication in Allium fistulosum chromosomes during S-phase.

Bromodeoxyuridine pulse labelling and immunodetection were applied to synchronized Allium fistulosum cells to study sequential changes in the chromosome replication pattern during S-phase. The replication patterns were classified into five main types depending on the location of the replication signals: (1) over the whole chromosomes; (2) at proximal and interstitial regions; (3) at proximal, interstitial and distal regions; (4) at interstitial and distal regions; and (5) at distal regions. The frequencies of each pattern changed sequentially according to the timing of BrdU incorporation, demonstrating the temporal order of chromosome replication during S-phase. The distal regions that correspond to the major C-bands replicated throughout S-phase except for the earliest stage, but most intensely in late S-phase. The replication time of different chromosome sites overlapped, which is quite different from the biphasic mode of replication that occurs in mammalian chromosomes.