Worm Breeder's Gazette 10(3): 34

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.

Cloning of C. elegans Homeobox-Containing Genes

Thomas R. Burglin and Gary B. Ruvkun

Figure 1

In an attempt to identify genes that control aspects of C.  elegans 
development, we decided to clone genes with homology to known 
developmental genes from other organisms, such as Drosophila.  We 
choose to clone homeobox-containing genes for two reasons: 1) 
Essentially all known Drosophila homeobox-containing genes are 
involved in control of development, thus we assume the same for C.  
elegans homeobox-containing genes.  2) The availability of a large 
variety of sequenced homeoboxes allowed us to define a well conserved 
8 amino acid region in the carboxy-terminus of the homeodomain (
Burglin, Cell 53, 339, 1988).
To detect the presence of this most conserved homeodomain region in 
C.  elegans, regardless of codon usage, we synthesized three 1024-2048 
fold degenerate oligonucleotides corresponding to two homeobox 
consensus types in the most conserved 8 amino acids.  Two of these 
degenerate oligonucleotides (PRD-1, PRD-2) code for the Drosophila 
homeobox consensus type derived from genes of the prd class.  The 
third degenerate oligonucleotide (HB-1 probe) was designed to detect 
all other Drosophila homeobox-containing genes known at that time (
Antp, eve, en, bcd classes), if one mismatch is allowed in the 
hybridization.  Control Southern blots of cloned Drosophila homeobox-
containing genes (eve, Dfd, prd, en, cad) showed that under our 
conditions the oligonucleotides of each type specifically hybridized 
to the corresponding homeobox-containing fragments and not to vector 
or other DNA sequences.
We screened the MIT cosmid library with a mixture of these probes 
and obtained about 100 clones which hybridized with varying intensity. 
DNA was isolated from these cosmids, digested with EcoRI, and 
analyzed by Southern blotting, independently with each type of probe.  
More than fifty of these cosmids showed one hybridizing band with the 
HB-1 probe, and several others showed specific hybridization with the 
PRD-1/PRD-2 probes.  Some clones showed two bands, suggesting the 
presence of homeobox gene clusters, as found in other organisms.  
Clones were sorted according to hybridizing fragment size and 
restriction pattern into about 45 groups.  Alan Coulson and John 
Sulston fingerprinted almost all cosmid clones detected with all three 
homeobox probes and mapped the true positives to 42 different contigs, 
with some contigs having several independent putative homeobox-
containing genes.  These results are not unexpected, since genomic 
Southern analysis using these oligonucleotides as probes revealed 
about 35-40 hybridizing bands.
In one case the correspondence between a putative homeobox-
containing cosmid and a known gene was found immediately.  Several 
overlapping cosmids we detected had previously been shown by Gwen 
Freyd to contain the lin-11 gene.  By hybridizing the HB-1 probe to 
lin-11 cDNAs (see article by Freyd, Kim and Horvitz) the positive 
hybridization of the cosmids was shown to be located within the lin-11 
gene.  Sequencing revealed that it has a true homeobox (Freyd, 
personal communication).
So far with have obtained sequence information from 2 of the new 
loci and confirmed that they contain a homeobox (see Figure).  We 
propose to name new homeobox-containing genes that cannot be assigned 
to any known gene 'Cehb' for C.  elegans homeo-box and we would like 
to suggest that they be numbered sequentially.  Hopefully this will 
prevent the confusion of nomenclature that arose in vertebrate 
organisms.  We have obtained only partial sequence information for the 
two new homeobox-containing genes Cehb-2 and Cehb-3, because both 
contain introns in the homeobox (indicated by the 'delta') and in Cehb-
3 there is a natural EcoRI site, which prevented further sequencing of 
this subclone.  Interestingly the intron in the homeobox of Cehb-2 is 
at the same position as one of the introns in JM#L1001 (=Cehb-1?; WBG, 
Vol.10, No.  2) and one of the introns in the Drosophila gene labial.
In conclusion, with three out of the three hybridizing loci 
sequenced, the odds are very favorable that most of the other loci 
will contain homeoboxes.  Can we expect to find even more homeoboxes?  
About 50% of the loci were only recovered as one clone, and we did not 
detect mab-5 (WBG, Vol.10, No.  2) and mec-3 (which we expected to 
detect), thus our screening appears not to have been exhaustive.  In 
addition, new classes of homeoboxes, like the unc-86 class (see 
article by Finney and Ruvkun), and the cut class, are so divergent 
from the other classes that we wouldn't (and didn't, in the case of 
unc-86) detect them with our oligonucleotides.  So we would expect 
that there are more homeobox-containing genes awaiting discovery.
[See Figure 

Figure 1