Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized.

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Differences in the localization and morphology of chromosomes in the human nucleus

DNA REPLICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 TRANSCRIPTION AND PRE-mRNA PROCESSING .... . . ) ....... ,.... 279 Localization of Transcriptionally Active Nuclear Regions by [ HJ-Uridine Incorporation or In Situ Nick Translation . . . . . . . . . . . . . . . . 279 Pre-mRNA Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 An Arginine/Serine-Rich Domain Targets Proteins to Nuclear Speckles . . . . . . 284 Effect of RNA Polymerase II Transcription on the Localization of Splicing Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,.... 285 Distribution of hnRNP Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Localization of Splicing Factors in the Amphibian Germinal Vesicle . . . . . . . . 289 Localization of Splicing Factors in Yeast 290 Distribution of Poly(A)'t RNA . . . . . . ... . . . . ..... . ..... . . . . .... 291 Distribution of Specific Cellular Transcripts . . . . . . . . . . . . . . . . . . . . . . . 291 NUCLEOLAR ORGANIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Macromolecular domains within the cell nucleus.

In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contrasts with expectations from the classical DSB repair model originally proposed for yeast meiotic recombination, but is consistent with models in which recombination is coupled intimately with replication. These results may explain why cytologically observable sister chromatid exchanges are induced only weakly by DNA-damaging agents that cause strand breaks, since most homologous repair events would not be observed. A preference for non-crossover events between sister chromatids suggests that crossovers, although genetically silent, may be disfavored for other reasons. Possibly, a general bias against crossing over in mitotic cells exists to reduce the potential for genome alterations when other homologous repair templates are utilized.

/pdf/sister-chromatid-gene-conversion-is-a-prominent-double-bxlwd2bg5x.pdf

Sister chromatid gene conversion is a prominent double‐strand break repair pathway in mammalian cells

Role of Chromosome Territories in the Functional Compartmentalization of the Cell Nucleus

The ura4+ gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4+ expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ura4+ gene adjacent to telomeres and at the three centromeric sites tested. The clr4-S5 and rik1-304 mutations cause the most dramatic derepression at two out of three sites within cen1. All six mutations had only slight or intermediate effects on a third site in the center of cen1 or on telomeric repression. Strains with lesions at the clr4, rik1, and swi6 loci have highly elevated rates of chromosome loss. We propose that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere.

/pdf/mutations-derepressing-silent-centromeric-domains-in-fission-48tmv3uqa2.pdf

Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.

Fluorescent in situ hybridization with chromosome specific probes was used in conjunction with laser scanning confocal microscopy to assess the three-dimensional distribution of chromosomes in human T-lymphocyte nuclei. Cells in the G1-phase of the cell cycle exhibit a distinctly non-random chromosome organization:centromeric regions of the ten chromosomes examined are localized on the nuclear periphery, often making contact with the nuclear membrane, while telomeric domains are consistently localized within the interior 50% of the nuclear volume. Chromosome homolog pairing is not observed. Transition from the G1 to G2 cell cycle phase is accompanied by extensive chromosome movement, with centromeres assuming a more interior location. Chromosome condensation and chromatin depleted areas are observed in a small subset of G2 nuclei approaching mitosis. These results demonstrate that dynamic chromosome rearrangements occur in non-mitotic nuclei during the cell cycle.

Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei.

https://www.cell.com/cell/pdf/0092-8674(92)90431-B.pdf

In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks One difference is in the location of maxicircles in situ In C fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk In contrast, T brucei maxicircles generally fill the entire disk Unlike those in C fasciculata, T brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts T brucei and C fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network In C fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment In T brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T brucei

/pdf/kinetoplast-dna-replication-mechanistic-differences-between-30jgc4iddp.pdf

Martin Ferguson

Papers

Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei.

In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication.

Kinetoplast DNA replication: mechanistic differences between Trypanosoma brucei and Crithidia fasciculata.