This grant addresses the interactions between homologous chromosomes during meiosis in yeast. Part I. Homolog Juxtaposition. (A) Mechanism of pre-DSB homolog pairing: We propose to identify (1) pairing-defective mutants using our Cre-loxP recombination assay and (2) pairing """"""""hot spots"""""""" using our high resolution FISH method. We think that the job of inter-homolog connection passes sequentially from one molecular process to another; this will be investigated by FISH and immunocytology of relevant mutants. Part II. Recombination and its Control. (A) We will continue theoretical analysis of crossover control, which will isolate mutants in which that control is """"""""extra robust,"""""""" and further analyze the chromatid interference we recently discovered. (B) We will further analyze a new recombination intermediate, single end invasions, with regard to detailed structured and functional dependencies; the timing of heteroduplex DNA formation will be investigated further; and we will analyze purify and analyze in detail, by gel and EM methods, double Holliday junctions from a single recombination hot spot, from wild type and Zipl mutant strains. (C) We will investigate the role(s) of synaptonemal complex component Zipl by exploiting our newly-discovered Zip1-dependent, temperature-dependent arrest condition to study a critical transition point; we will also isolate thermosensitive Zip1 alleles and use them to define time(s) of Zipl action by temperature shift experiments; certain special types of zipl alleles will also be isolated; and the localization of leptotene Zip1 patches relative to other proteins will be assessed cytologically. (D) We will investigate the roles of Mlhl and related proteins, which likely define new functions, by (1) genetic and physical analysis of recombination in relevant mutants and (2) biochemical analysis aimed at identifying and characterizing Mlhl-containing protein/protein and protein/DNA complexes. Part III. Chromosome/Chromatin Structure. (A) We will exploit our newly developed method, C-SCAN, which determines, in a single experiment, the distribution of a chromosomal protein of interest all along the length of Chromosome III. We will continue analysis of Red1 (and its relative Hop1) and will also analyze Zipl, selected other meiosis-specific proteins and several basic components of chromosome structure. (B) We think that meiotic prophase involve multiple cycles of chromatin compaction and decompaction, certain of which are critical for recombination. We will further investigate chromosomal protein Bdf1 and variations in histone status in this regard by genetic, C-SCAN and other methods. Supercoiling status of loops may be addressed.
Showing the most recent 10 out of 62 publications
