Direct evidence for interphase chromosome movement during the mid-blastula transition in Drosophila.

In Drosophila, several genetic phenomena are most easily explained by a model in which homologous chromosomes pair, at least transiently, and use regulatory information present on only one homolog to pattern expression from both homologs [1] [2] [3]. To accomplish pairing of sites on different chromosomes, there must be a mechanism by which communication between homologs is facilitated. However, except in the case of meiotic prophase, directed, rapid chromosomal movement has not yet been observed. Some studies suggest that chromosomes are relatively immobile during interphase [4] [5], while others suggest that chromatin can reposition during interphase [6] [7] [8] and may be free to undergo substantial Brownian motion [9]. Using high-resolution, three-dimensional imaging techniques, we determined directly the structure and nuclear location of eleven different loci, both active and inactive, in embryos at cycle 14, the mid-blastula transition. We show that during a single interphase, portions of chromosomes moved in a cell-cycle-specific, directed fashion, independently and over long distances. All eleven regions showed movement, although the genes closer to the centromere moved faster (0.7 microm/minute) and over long distances (5-10 microm), whereas those nearer the telomere expanded in the same place and became oriented along the nuclear axis. Gene motion was independent of replication, transcription and changes in nuclear shape. Because individual genes on the same chromosome move independently, the movement is unlikely to be mediated by centromeres, Brownian motion or random drift and must be caused by an active mechanism.

'Chromosomal puffing' in diploid nuclei of Drosophila melanogaster.

In situ hybridization has become a powerful technique for dissecting nuclear structure. By localizing nucleic acids with high precision, it is possible to infer the native structure of chromosomes, replication factories and transcript processing complexes. To increase the value of this technique, we have established the limits of resolution of two-color in situ hybridization to chromosomal DNA in diploid chromosomes of Drosophila embryonic nuclei. Using high-resolution 3-dimensional optical microscopy and computational image analysis, we establish that we can distinguish the location of chromosomal sequences that lie 27-29 kb apart within a 40 kb transcription unit with an accuracy of about 100 nm. Contrary to observations made in mammalian tissue culture cells, we find that when the gene is expressed, it assumes an open configuration, and that the extent of decondensation is variable from chromosome to chromosome. Further experiments suggest that variation in gene structure results from asynchrony in transcriptional elongation. We suggest that the phenomenon we observe is the diploid analog to chromosomal puffing that occurs in the transcriptionally active regions of Drosophila polytene chromosomes.

The onset of homologous chromosome pairing during Drosophila melanogaster embryogenesis.

We have determined the position within the nucleus of homologous sites of the histone gene cluster in Drosophila melanogaster using in situ hybridization and high-resolution, three-dimensional wide field fluorescence microscopy. A 4.8-kb biotinylated probe for the histone gene repeat, located approximately midway along the short arm of chromosome 2, was hybridized to whole-mount embryos in late syncytial and early cellular blastoderm stages. Our results show that the two homologous histone loci are distinct and separate through all stages of the cell cycle up to nuclear cycle 13. By dramatic contrast, the two homologous clusters were found to colocalize with high frequency during interphase of cycle 14. Concomitant with homolog pairing at cycle 14, both histone loci were also found to move from their position near the midline of the nucleus toward the apical side. This result suggests that coincident with the initiation of zygotic transcription, there is dramatic chromosome and nuclear reorganization between nuclear cycles 13 and 14.

Optical sectioning and three-dimensional reconstruction of diploid and polytene nuclei.

To build a coherent picture of mitosis and cell fates during blastoderm and through the complex movements of gastrulation, it will be important to localize and follow several markers simultaneously in live specimens, ideally in 3D, using high-resolution, specific, noninjurious staining and observation procedures. The study of early Drosophila development has already profited from the use of fluorescent labeling and low-light-level imaging of live embryos using a CCD camera. Chromosomes in fixed samples have been labeled using DNA-specific dyes, making the pattern of mitotic patches visible. In vivo, 3D microscopy of fluorescently tagged chromosomes, in conjunction with computerized image processing, has permitted the first direct cell lineage analysis in the early Drosophila embryo. Moreover, the techniques adapted to study Drosophila development have been used for analysis of Drosophila chromosome structure, mitosis, and cell cycle, and are general enough to be applied to a myriad of problems in cell biology. "Optical sectioning" has always been used to scrutinize everything from onion roots to frog eggs, focusing up and down through the specimen, with the observer's brain responsible for the image processing. However, the volume of raw data generated by the high-resolution approach detailed above requires the use of sophisticated and adaptable computer systems to analyze and organize the results. Software designed to extract information from these complex images, either automatically or through an interactive approach, will become essential tools for cell and developmental biology. The brain of the experimenter remains the most important component in any image-processing system, but the support of technology will be essential.

Precise determination of the molecular limits of a polytene chromosome band: regulatory sequences for the Notch gene are in the interband.

We have aligned the molecular map of the Notch locus to the cytological features of the salivary gland polytene chromosomes of D. melanogaster in order to determine the interphase chromatin structure of this gene. Using high-resolution in situ hybridization and computer-aided optical microscope data collection and image analysis, we have determined that the coding portions and introns of the Notch gene, which is not expressed in this tissue, are all contained within the polytene chromosome band 3C7. The portion of the Notch gene that resides 5' to the start of transcription lies in an open chromatin conformation, the interband between bands 3C6 and 3C7. Our data are most consistent with condensation of the chromosomal DNA into 30 nm fibers in this polytene band.

Human genome organization: Alu, lines, and the molecular structure of metaphase chromosome bands.

Combining high resolution in situ hybridization with quantitative solid state imaging, we show that human metaphase chromosome Giemsa/Quinacrine and Reverse bands are each characterized by distinct families of interspersed repeated sequences: the SINES, Alu family dominates in Reverse bands, and the LINES, L1 family dominates in Giemsa/Quinacrine positive bands. Alu is 56% guanine plus cytosine, and L1 is 58% adenine plus thymine, and each may comprise 13%-18% of the total DNA in a chromosome band. Therefore, the distribution of these sequences alone may account for a large part of human chromosome banding seen with fluorescent dyes. With the exception of some telomeric regions, and the chromosomal regions of simple sequence DNA, Alu and L1 are precisely inversely distributed, suggesting an inverse functional relationship. This finding links genome organization with chromosome structure and function.

Histone H2A subtypes associate interchangeably in vivo with histone H2B subtypes.

The yeast Saccharomyces cerevisiae contains two primary sequence subtypes of histone H2B (H2B1 and H2B2) and of H2A (H2A1 and H2A2). Mutants in each of the H2B subtypes have been used to show previously that yeast cells lacking one or the other, but not both, of the H2B proteins are viable. Because H2A protein interacts in the nucleosome with H2B, we wished to determine whether specific H2A subtypes must interact with specific H2B subtypes. We describe experiments in which frameshift mutations were introduced into both of the H2A genes in vitro and the mutant genes integrated into the yeast genome, replacing the wild-type H2A genes by a subsequent recombination. Using these mutant (hta1- and hta2-) strains we find that neither H2A gene has a unique essential function during any phase of the yeast life cycle, although strains homozygous for hta1- grow more slowly. However, one functional H2A gene is required for viability because cells mutant in both H2A genes arrest at spore germination prior to bud separation. By combining these H2A mutations with the H2B mutations obtained previously, we show that all combinations of H2A and H2B subtypes produce viable cells. From these genetic experiments and electrophoretic analysis of the histone proteins of these mutants we conclude that the H2A subtypes can associate interchangeably with the H2B subtypes.