The structure of higher eukaryotic chromosomes

Chromosomal structure has been analyzed from the standpoint of core structure and the relationship between interphase and metaphase chromosomal forms. A possible relationship between prokaryotic and eukaryotic chromosomes has also been proposed. The general structural plan offered is a series of DNA-histone loops extending laterally from a core held together by disulfide bond linkages. The models proposed have been derived from a loop model with core very recently proposed by H. Sobell. A short experimental section of this paper demonstrates that S-S cleaving agents as well as trypsin cause easily observable effects on human metaphase chromosomes.     

Replication origins in yeast chromosomes

DNA replication initiates at many sites in eukaryotic chromosomes. It has been difficult to isolate such replication origins, but a family of sequences from the yeast genome have properties which suggest that they may serve this function. The identification of these sequences together with sophisticated methods of genetic analysis, make yeast a useful organism for the study of eukaryotic DNA replication.     

Folded chromosomes in non-cycling yeast cells

Folded chromosomes from stationary phase or ammonia-starved yeast (Saccharomyces cerevisiae) cells can be isolated as compact structures, distinct and separable by sedimentation from the folded chromosomes of pre-replicative (G₁) and post-replicative (G₂) nuclei. Such cells are in a dormant or non-cycling (G₀) stage. The folded genome from such cells is referred to as theg ₀ form and has a sedimentation velocity of about 1700S. Sedimentation analysis of mixed G₀ and G₁ and G₂ lysates indicates that theg ₀ structure is not an artifactual breakdown product of theg ₁ org ₂ structures. A comparison of the proteins fromg ₀ versusg ₁ andg ₂ structures by gel electrophoresis has revealed differences in about 10–11 non-histone and perhaps 2 histone proteins. Entry into the G₀ stage, and emergence into G₁ after G₀ arrest, are accompanied by an ordered transition fromg ₂ tog ₁ tog ₀, and fromg ₀ tog ₁ tog ₂ forms, respectively. Hence, entry into G₀ and re-emergence from G₀ can be considered as differentiative processes, not normally part of the cell cycle, and accompanied by specific changes in the tertiary organization of the genome.     

Recognition of specific DNA sequences in eukaryotic chromosomes.

The packaging of DNA into chromatin probably places certain restrictions on how specific DNA sequences can be recognized by DNA sequence specific recognition proteins (SRP). Several unique features of this type of interaction are discussed. Specifically, as a consequence of the coiling of the DNA about a histone core, it is proposed that DNA recognition sites will be compound and that each element of the compound recognition site will be about 10 - 20 b.p. in length and distributed at approximately 80 b.p. intervals--the periodicity of the DNA wrapping around the nucleosome.     

Detection of cryptic bands by AluI in eukaryotic chromosomes

Selective digestion of fixed chromatin with the restriction endonuclease AluI (which cuts the sequence AG CT) uncovers a specific and repeatable pattern of bands within the euchromatin of two species of grasshoppers and of the L929 mouse cell line, which are not detectable by means of other banding techniques such as C-bands, specific fluorochromes, or other restriction endonucleases. It is tentatively suggested that this chromatin represents a special class of repetitive DNA embedded in the euchromatin, not containing the AluI restriction site to the same extent as in euchromatin and not associated with C-banded heterochromatic material.     

Behavior of dicentric chromosomes in budding yeast

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.     

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.     

Double-strand breaks on artificial chromosomes in yeast

Yeast artificial chromosomes composed primarily of bacteriophage λ DNA exhibit very low levels of meiotic crossing over compared with similarly sized intervals of natural yeast DNA. When these recombinationally quiet chromosomes were augmented with a 12.5 kb insert of sequences from yeast chromosome VIII, genetic studies demonstrated that the artificial chromosomes had acquired recombination properties characteristic of this region of chromosome VIII. On authentic yeast chromosomes, most meiotic recombination events are initiated at sites where the DNA is cleaved to create a double-strand break (DSB). This report describes physical analyses that were carried out to examine the relationship between DSB sites and the recombination behavior of the artificial chromosomes. The results show that DSBs are rare on these artificial chromosomes, except for the 12.5 kb insert. Mapping of the DSB sites shows that their positions correlate with the previously determined positions of DSB sites on chromosome VIII. Deletion of two characterized chromosome VIII DSB sites from the 12.5 kb insert on the artificial chromosome resulted in the loss of the predicted DSB fragments and a reduction in crossing over between artificial chromosomes. Received: 15 May 1998; in revised form: 26 September 1999 / Accepted: 18 November 1999     

Radiation-induced instability of yeast chimeric chromosomes

Instability of the I chimeric chromosome of the yeast Saccharomyces induced by gamma-irradiation has been studied. The chimeric chromosome analysed contained an integrated pYF91 plasmid. Cells of the integrant were irradiated and then mated with non-irradiated cells of the proper tester strain marked by ade1 mutation (red colour of colonies). We isolated 10 hybrids with pink colonies on selective medium. They displayed high degree of mitotic instability during growth on nonselective medium, segregating red colonies (15 to 90% of the total). Tetrad analysis showed that some of the unstable chromosomes exhibited lethal effect in haploids, while others were viable and could pass through meiosis retaining their instability.     

酿酒酵母染色体设计与合成研究进展

随着合成生物学的蓬勃发展,基因组学的研究正在由读取基因组信息拓展到以编写基因组信息为主的合成基因组学时代。2009年,由Jef D. Boeke教授提出的人工合成酵母基因组计划(Sc2.0)旨在合成世界上首个真核生物基因组。在美、中、英、法、澳大利亚、新加坡等多国科学家的努力下,目前已经完成1/3的酵母染色体的人工合成。本文从合成基因组学领域的发展历程出发,介绍了Sc2.0计划中酿酒酵母(Saccharomyces cerevisiae)染色体设计与合成的最新进展,包括酿酒酵母9号染色体右臂、3号染色体、2号染色体、5号染色体、6号染色体、10号染色体和12号染色体的设计与合成过程,阐述了其各自的合成策略以及生物学意义,以期为合成基因组学的深入开展提供借鉴与参考。     

Hierarchy of organization in eukaryotic chromosomes (a review)

Summary Several models of macromolecular arrangements in eukaryotic chromosomes have been proposed during the past fifteen years. Many of the models are consistent with physical and chemical data on the molecular components of chromosomes, and a few have the appearance of meeting the requirements for cytological organization in chromosomes. However, one of the most frustrating problems in developing a working model is to provide a scheme that fits genetic function while satisfying the structural parameters. This has not yet been achieved.Although emphasis in this review has been placed on uninemic and polynemic models, alternatives, such as a bineme, for example, remain. It is clear, moreover, that the issue can be resolved only through continued efforts to make direct observations of chromosomes with light and electron microscopy coupled with the additional tools ofX-ray analysis and analytical biochemistry. A recent analysis byWray andStubblefield (1969) has led to a rather innovative model of the chromosome, and exemplifies the kind of approach needed to clarify the phenomenon. Furthermore, analyses of meiotic chromosomes may provide valuable insight for relating organization to genetic function (cf Maguire, 1966 andBraselton, pers. comm). Of particular interest are mutation events as related to subchromatid organization, and the reorganization of chromosomal fibrils during early meiotic stages. At present, and as a generalization, the evidence points more strongly toward at least a binemic arrangement of chromosomal subunits than toward a uninemic one.     

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.     

Loop organization of eukaryotic chromosomes and triple-stranded DNA structures

To study possible involvement of polypurine and polypyrimidine DNA tracts capable of forming triple-stranded structures (the H-form of DNA) in compaction of eukaryotic chromosomes, an in silico search for complementary polypurine and polypyrimidine sequences was carried out within 12 eukaryotic genes. It was shown that, in chromosomal gene loci, 10–11 bp polypurine and polypyrimidine tracts potentially capable of interacting with each other with the formation of triplex structures (“structuring” regions) are located in predominantly in introns and gene-flanking regions. In vivo, such DNA-DNA interactions can result in the chromosomal gene domain folding into several small loops. The character of the DNA triplex-mediated compaction of chromosomal gene loci may be related to gene functioning. A similar analysis of long (LINE) and short (SINE) interspersed repeat sequences, as well as of satellite DNA, showed essential resemblance between the compaction mechanisms of coding and noncoding chromosome regions.     

Telomeres: the beginnings and ends of eukaryotic chromosomes

The ends of eukaryotic chromosomes are called telomeres. This article provides a short history of telomere and telomerase research starting with the pioneering work of Muller and McClintock through the molecular era of telomere biology. These studies culminated in the 2009 Nobel Prize in Medicine. Critical findings that moved the field forward and that suggest directions for future research are emphasized.