This is a request for four years of support to investigate mechanisms by which meiotic prophase homologous chromosomes engage in synapse. The proposal utilizes the mouse as the experimental organism and relies on multicolor FISH studies as well as fluorescence-based antibody detection of key proteins which may be involved.
Three specific aims are proposed.
Aim 1 will address the homology search process. Using combinatorial labeling and 21-color display, the locations of homologous chromosomes relative to one another in premeiotic S phase will be determined. Their movements through synapsis in meiotic murine prophase will be determined. The relationship between the degree of chromosome condensation and association at synapsis of homologues will also be determined. This will be accomplished by computer analysis of the images where the length of the chromosome, the amount of association along the total chromosome length, and points of contact relative to the distal end will be measured. Ratios of these values should give an indication of condensation and association. Finally, these studies will be combined with antibody detection of Rad51, a RecA homologue which appears to play a role in gametogenesis.
Aim 2 will attempt to distinguish between two leading models of synaptic initiation. The gene conversion model involves double-strand breaks which by interaction with a RecA molecule, form a presynaptic filament which facilitates sequence recognition and triplex formation. There is evidence in support of this model in yeast, and this model predicts repair synthesis. In contrast, a preselection model, for which evidence is found in lilies, suggests that delayed replication occurs at certain regions along the chromosome (GC-rich subfraction in lilies). This model predicts semi-conservative replication. Several approaches will be used to test these models. In one, the relative appearance of double-strand breaks in relation to the appearance of Rad51 will be assessed. This will be accomplished using anti-ku, labeled with one fluor, which recognizes broken chromosome ends and anti-Rad51, with another fluor. Co-appearance supports the gene conversion model while delayed appearance of ku would support the preselection model. Similar experiments using antibody against polymerases thought to be involved in replication vs repair will be used to support these findings. Finally, chromosome aberrations will be used to ask the influence of nonhomologous synapsis on Rad51 foci in addition to determining if DNA synthesis occurs using BrdU incorporation. Semi-conservative replication would be considered support for the preselection model.
Aim 3 will define factors which effect preselection of sequence. Here the importance of the timing of replication vs the sequences themselves on the selection of Rad51 associated sequences. DNA sequences linked to genes exhibiting suppression will be characterized and those mapping to R-bands will be used for replication timing experiments. Early and late replication control sequences as well as the above test sequence will be combinatorial-labeled and tested on spermatocytes of normal and Robertsonian translocations.
Other aims here will be to examine the temporal expression of other proteins that may be involved in synaptic initiation including FBP which can promote localized melting.
| Froenicke, Lutz; Anderson, Lorinda K; Wienberg, Johannes et al. (2002) Male mouse recombination maps for each autosome identified by chromosome painting. Am J Hum Genet 71:1353-68 |
