Chromosome walking and gene cloning using a Neurospora crassa Linkage Group VI-specific library.
Thomas J. Schmidhauser - Department of Biology, University of Southwestern Louisiana,
Fungal Genetics Newsletter 46:22
Chromosome walks from the cpc-1 and Bml loci were extended and the cys-2 locus cloned by sib-selection using a subset of the Orbach/Sachs Neurospora crassa genomic library.
One new step has been taken from one end of the Bml walk using a cosmid G12:10:C based probe (Figure 1). No new cosmids have been found to extend the other end of the Bml walk or of the cpc-1 walk. Several additional overlapping cosmids were identified in the extension of the cpc-1 walk. The X24:12:C probe also identified cosmids G8:8:C and G9:1:B, the X25:9:C probe also identified cosmid X9:12:G. We have not determined the size of the additions to the cpc-1 and Bml walks.
The LGVI library was divided into 28 primary pools by pooling cosmid DNA representing one or two rows from each microtiter dish. Each primary pool has from six to twelve cosmid clones.
Two rounds of transformation into cys-2 spheroplasts identified a
cys-2+ cosmid, X11:5:C. A probe based on X11:5:C identified one
additional cosmid, X17:4:F, which is also cys-2+ (Figure 1). Cloning
of cys-2+ establishes that the walk from the cpc-1 locus
towards the LGVIL telomere has not reached the cys-2 locus (Figure 1).
Figure 1. Chromosome walks on Neurospora LGVI. The figure is not to scale.
(A) Seven steps added to the cpc-1 walk. (B) Three cosmids identified using a G12:10:C probe from the Bml walk. (C)Two cys-2+ cosmids. (D) Relative locations of the cpc-1 and cys-3 walks on LGVIL.
The location of the Bml walk on LBVIL has not been established with respect to the cpc-1 walk.
Return to the FGN 46 Table of Contents
Return to the FGSC main page