Worm Breeder's Gazette 16(1): 44 (October 1, 1999)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Closing in on mut-2: physical mapping of let-86 via cosmid rescue.

Queta Boese , John Collins

Department of Biochemistry and Molecular Biology, UNH, Durham, NH 03824

In our ongoing effort to clone mut-2, we have characterized a temperature-sensitive sterile phenotype conferred by mut-2(r459).  Knowing that mut-2 maps between dpy-14 and sem-4 on LG I, we tested two small free duplications that bisect this region for rescue of ts sterility. hdp65 rescues; hDp62 does not (see fig 1.)  This puts mut-2 between let-86, which defines the left end of hDp65, and sem-4 on the genetic map. To define the corresponding physical interval we wanted to position let-86 on the physical map. sem-4 has been cloned: it is contained on cosmid E02H2, which overlaps H06O01 at the right end of the mut-2 interval.

let-86 confers a larval lethal phenotype; it is maintained as a wild type heterozygote (BC244: genotype: dpy-14 let-86+unc-13/ ++unc-15+).  BC244 hermaphrodites produce WT het (50%), Unc (25%) and  Dpy Unc (25%) progeny.  The DpyUncs die as L1s.  To position let-86 physically, we tested cosmids T10B11, C30F12 and H06O01 for the ability to rescue let-86. To establish transgenic lines, each was microinjected into N2 adults as a complex mix along with a rol-6(su1006) clone and linearized N2 DNA. The arrays were then crossed into the let-86 background and screened for rescue (viable Dpy Unc progeny). Animals transgenic for C30F12 segregated viable Dpy Uncs; animals transgenic for T10B11 and H06O01 segregated Dpy Uncs that died asL1 larvae.  This defined C30F12 as the cosmid containing let-86.  C30F12 contains 45kb of C. elegans DNA, and seven predicted ORFs.  Five of the seven ORFs are novel, one has homology to a seven transmembrane receptor (C30F12.6) and another is similar to isocitrate dehydrogenase (C30F12.7).

Rescue of let-86 by C30F12 establishes that mut-2 is on either C30F12 or H06O01 (mut-2 is between let-86 and sem-4 -- Figure 1).Microinjection of H06O01 rescues the ts sterile phenotype of mut-2.This cosmid contains four ORFs, including H06O01.2, which encodes a homologue of CHD1 (chromodomain-helicase-DNA binding protein). Considering the multiple roles of mut-2 in germline chromatin and chromosome dynamics, we consider this to be the best candidate at present. Current efforts are focused on testing this ORF for rescue and identifyng the molecular lesion in r459 ....... and so the saga continues!

1) Boese, Q and J. Collins. 1999. IWM:197