Diamond professorial fellowship for imaging chromosomes by coherent X-ray diffraction

The chromosome is the repository of all genetic material in eukaryotes. Humans have 22 separate chromosomes in their karyotype plus the special X/Y sex-determining chromosome. One third of the life cycle of the chromosome, metaphase, is involved with mitosis, whereby the safe transmission of all the genetic material to progeny cells is undertaken; the remainder of the time, interphase, is when the genes are are unpacked and transcribed to operate the cellular machinery and copied to make the daughter chromosomes. Mitosis is an elaborate combination of cellular processes of which the protective packaging of the DNA into safe organized bundles - the chromosomes - is one of the fundamental steps. The first levels of organization of chromatin, the double helix, the nucleosome (DNA-histone protein complex) and the 'beads on a string' (BoS), packed into 30nm fibres with histone H1, have represented major breakthroughs in biology resulting in Nobel prizes. These levels of structure, summarized in Fig 1, are now relatively well understood, although there are still several possible models discussed for the fibre, and possibly some interesting diversity also. It is the next level of structure of the metaphase chromosome where the organisation becomes complicated and the research proposed here will begin to have impact. To protect the genes in transit though mitosis, they are packed together tightly into the familiar X-shaped chromosome pairs that separate once the cell division begins. The 30nm fibres are presumably coiled up in a regular superstructure at the next level within the chromatids; this coiling is the structure we intend to image by X-ray methods. This packing is achieved by scaffolding proteins which also protect the DNA mechanically from forces that could damage the genes. The optical refractive index of this dense complex is high enough that chromosomes are most easily visualized in metaphase. The high density also means that metaphase chromosomes can be handled under the optical microscope with micromanipulation tools that are familiar to cytogeneticists. We plan to use these same tools for sample preparation for the X-ray imaging experiments. This proposal plans to undertake a full 3D imaging of the chromosome at the 30nm resolution level. The metaphase chromosome is of necessity a compact object devoid of hanging strands or loops that would interfere with mitosis. This works well with the use of a 'support' constraint in phasing methods partly developed by the PI. The chomosome electron density is high for a biological substance because of the tightly-packed phosphorus (and counterions associated with the DNA). Staining methods might even be developed to enhance the X-ray contrast. The well-defined boundary will enable support-seeking methods such as 'shrinkwrap' to work effectively. Because they are readily manipulated, as described above, individual chromosomes can be isolated and mounted on pins or fiducialised membranes for measurement. Yet the chromosome is not expected to be a highly reproducible structure like the ribosome or certain viruses that can be solved by MX or the newer method of serial crystallography with an X-ray free-electron laser. In fact, much of the interest in cytogenetics lies in the differences between chromosomes of individuals and between copies from the same individual. All this can be contemplated and attempted in the context of the HRC.