Cyanobacterial multi-copy chromosomes and their replication

ABSTRACT

While the model bacteria Escherichia coli and Bacillus subtilis harbor single chromosomes, which is known as monoploidy, some freshwater cyanobacteria contain multiple chromosome copies per cell throughout their cell cycle, which is known as polyploidy. In the model cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, chromosome copy number (ploidy) is regulated in response to growth phase and environmental factors. In S. elongatus 7942, chromosome replication is asynchronous both among cells and chromosomes. Comparative analysis of S. elongatus 7942 and S. sp. 6803 revealed a variety of DNA replication mechanisms. In this review, the current knowledge of ploidy and DNA replication mechanisms in cyanobacteria is summarized together with information on the features common with plant chloroplasts. It is worth noting that the occurrence of polyploidy and its regulation are correlated with certain cyanobacterial lifestyles and are shared between some cyanobacteria and chloroplasts.     

Light-dependent dimerisation in the N-terminal sensory module of cyanobacterial phytochrome 1

Phytochromes, photoreceptors controlling important physiological processes in plants and many prokaryotes, are photochromic biliproteins. The red-absorbing Pr ground state is converted by light into the farred-absorbing Pfr which can be photoconverted back to Pr. In plants at least Pfr is the physiologically active signalling state. Here, we show that the N-terminal photochromic module of Cph1 homodimerises reversibly and independently in Pr and Pfr, Pfr-dimers being significantly more stable. Implications for the mechanism of signal transduction are discussed.     

Structure and replication of Phaseolus polytene chromosomes

The normal morphology of the polytene chromosomes of the embryo suspensor of Phaseolus coccineus is that of a tightly condensed cord with heavily Feulgen staining centromeric heterochromatic regions (α-heterochromatin) and other accessory heterochromatic regions (β-heterochromatin). The replication pattern of the chromosomes has been determined by autoradiographic analysis of material pulsed with ³H-thymidine for various lengths of time. The DNA replication cycle reqires 4–6 hours for completion. During replication chromosome structure becomes diffuse and the β-heterochromatic regions are indistinguishable from the euchromatic regions. The euchromatin is the first to replicate, and replication begins simultaneously at numerous sites in the euchromatin. The β-heterochromatin replicates next, and finally the centromeric heterochromatin. Replication is essentially complete in each of these parts of the chromosome before DNA synthesis begins in the next. The chromosomes are composed of numerous longitudinally running Feulgen positive strands, the equivalent portions of which replicate simultaneously. This indicates that there must be close control of the replication cycle in sister strands.     

A family of cosmid vectors with the multi-copy R6K replication origin

A family of cosmid vectors was constructed which contain replication origins (ori) derived from the multicopy plasmid R6K, a kanamycin resistance gene and two cos sites, permitting efficient library construction. Additional features of later constructs are (i) the presence of NotI sites flanking the site of insertion to allow intact excision of inserts, (ii) the facility for selective cloning of the ends of inserts for rapid chromosome walking, and (iii) the use of a mutated R6K ori leading to an increased copy number.     

Initiation of DNA replication in yeast chromosomes

A family of DNA fragments from the yeast genome has properties that suggest that chromosome replication starts at specific DNA sequences. These elements (autonomously replicating sequences: ARS) have a bipartite structure: a small (less than 20 base pairs) AT-rich region essential for function, flanked by larger regions important for maximal activity of the replicator. In an attempt to identify proteins involved in initiation of replication, yeast mutants that show an enhanced ability to replicate minichromosomes with defective ARSS have been isolated.     

Origin of replication in circular prokaryotic chromosomes

To predict origins of replication in prokaryotic chromosomes, we analyse the leading and lagging strands of 200 chromosomes for differences in oligomer composition and show that these correlate strongly with taxonomic grouping, lifestyle and molecular details of the replication process. While all bacteria have a preference for Gs over Cs on the leading strand, we discover that the direction of the A/T skew is determined by the polymerase-alpha subunit that replicates the leading strand. The strength of the strand bias varies greatly between both phyla and environments and appears to correlate with growth rate. Finally we observe much greater diversity of skew among archaea than among bacteria. We have developed a program that accurately locates the origins of replication by measuring the differences between leading and lagging strand of all oligonucleotides up to 8 bp in length. The program and results for all publicly available genomes are available from http://www.cbs.dtu.dk/services/GenomeAtlas/suppl/origin.     

Initiation of DNA replication in human chromosomes

DNA replication within the first 10 min of the S phase was studied using synchronized human diploid cells. It appeared that every chromosome in the human genome, including late-replicating X, had segment(s) which initiated DNA replication within the first 10 min of the S phase. The position, the shape and the size of these segments corresponded to those of Q(G)-negative bands suggesting that each of them constitutes a basic unit of initiation of DNA replication.     

X chromosome choice occurs independently of asynchronous replication timing

In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation.     

Analysis of DNA replication patterns of human fibroblast chromosomes the replication map

Summary A replication map of human fibroblast chromosomes from two diploid human female fibroblast lines, 46,XX and 46,X, del (X)(q13), was determined using the fluorescent plus Giemsa (FPG) technique. Each chromosome was found to stain homogeneously dark when thymidine was incorporated for the entire S phase of that particular cell. As the duration of exposure to thymidine progressively decreased by increasing the incubation time in bromodeoxyuridine, the staining intensity of chromosomes decreased and, concurrently, gaps in the staining began to appear. These gaps coincide with R bands and represent the earliest areas to complete DNA synthesis. As these areas widen and increase in frequency, first Q and G bands appear, and finally C bands.Homologous X chromosomes were easily differentiated by either a comparison of the bands present or their staining intensity. The replication kinetics of the structurally abnormal heterocyclic X chromosome were very similar to those of the normal heterocyclic X chromosome. The X chromosome with deletion of a portion of the long arm was consistently late in replication.     

A fractal model of chromosomes and chromosomal DNA replication

With the aim of clarifying topological problems involved in the process of chromosomal DNA replication, a fractal model of chromosomes was built based on the assumption that a part of a chromosome, e.g. a radial loop, is similar in shape to a whole chromosome and each radial loop represents structures in the lower-order organization (an assumption of self-similarity). Several other assumptions used include (i) one continuous DNA fiber makes a whole chromosome (a unineme hypothesis), (ii) in situ DNA exists in the form of a double duplex or a tetraplex which is made of two duplex DNAs, although a duplex DNA may appear transiently in S-phase (multi-strandedness hypothesis) and (iii) torsional stress on a DNA fiber causes the fiber to supercoil and thus stabilizes chromosome structure (torque-based stabilization). This model allowed to calculate of a fractal dimension of a representative metaphase chromosome (e.g. d = 2.34), to predict the mode of replication of double duplex and to furnish a topological basis for the decondensation unit hypothesis. It must also be admitted that all the arguments in this report except for the possible existence of split telomeres hold true without assuming a tetraplex organization of chromosomes. Implications of this model was discussed and the importance of the fractal dimension as a measure of chromatin condensation stressed.     

Mapping replication origins in yeast chromosomes.

The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.     

Insect sex chromosomes. IV DNA replication in the chromosomes of Gryllotalpa fossor

In Gryllotalpa the cell cycle duration in the hepatic caecae in vivo is about 12.5 h and of various phases are, G2 + P about 10 h, S about 2.5--3.5 h, and G1 appears negligible or absent. These estimates of the cell cycle are the only ones available in Gryllotalpidae. In the female Gryllotalpa, as in mammals, there is asynchronous DNA replication between the two euchromatic arms of the two X chromosomes. The other arm is constitutively heterochromatized and as expected is late replicating. Thus, a regulatory mechanism for dosage compensation by X chromosome inactivation appears to be operating in Gryllotalpa. This we believe, is the first cytogenetic demonstration of such a mechanism outside mammals.     

Heterochromatin and late replication in the chromosomes of the rat

The distribution of heterochromatin in the chromosomes of the rat was determined by analysing two of its properties: late replication and differential stain with the DNA d-r method. The presence of late and non-late replicating c-heterochromatin in the genome of Rattus norvegicus indicates that this chromatin is an heterogeneous substance exhibiting different properties. Furthermore, the existence of heterochromatin formed by nonrepeated sequences or by sequences with a low degree of repetitiveness is suggested by the presence of late replicating areas which do not react with the DNA d-r method.Supported by grants from the Conicet and Cic.