Worm Breeder's Gazette 8(3): 84

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.

The Cloning of the Region Around unc-22

K.A. Beckenbach, D.L. Baillie, A.M. Rose

Figure 1

The region around unc-22, flanked by unc-43 on the left and unc-31 
on the right, has been intensively studied in our laboratory over the 
last eight years using conventional genetic analysis.  We have now 
embarked upon a molecular analysis of this same region.  We have at 
present identified and recovered about 300 kb of DNA that lies within 
this two map unit interval.  Tc1 induced restriction fragment length 
differences closely linked to unc-22 were identified in the N2 and BO 
strains.  DNA was prepared from a strain constructed by serial 
backcrossing of N2/BO heterozygotes to N2 males (where the BO 
chromosome IV was marked with a EMS induced unc-22 allele).  After 
each cross the unc-22 marked BO chromosome was recovered homozygous.  
This procedure was repeated six times.  A lambda gt10 EcoRI library 
was constructed and subsequently screened for Tc1-containing sequences.
These phage were isolated and their genomic fragments subcloned into 
pUC13 or pUC19 plasmid vectors.  The flanking sequences were recovered 
by EcoRV digestion followed by blunt end ligation and recloning of the 
Tc1 deleted plasmid.  These plasmids provided convenient probes for 
the mapping of the associated N2/BO RFLD.  Three factor mapping was 
carried out using the marker pairs unc-43 
unc-31 to map the plasmid-defined RFLD.
The resulting newly-defined molecular sites are shown on the 
accompanying figure.
The plasmids which defined each site were then used to isolate 
genomic clones from our lambda Charon 4 library (prepared by T.  
Snutch).  About 25-30 kb of flanking sequence was recovered for each 
probe.  These lambda phage were in turn checked for overlap with 
existing cosmids in John Sulston's genomic mapping collection.
[See Figure 1]

Figure 1