Worm Breeder's Gazette 10(1): 21

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Isolation and Characterization of rol-6

R. French and J. Kramer

Figure 1

Our lab is interested in the interaction of collagen molecules in 
the assembly of the cuticle.  Since the cuticle is composed mainly of 
collagen, it is reasonable to assume many of the genes affecting 
cuticle morphogenesis will be collagen genes.  rol-6 is a 
developmentally regulated genetic locus affecting cuticle 
morphogenesis.  Mutations in rol-6 result in the formation of right 
handed, helically twisted cuticles.  There are dominant, recessive and 
null alleles of rol-6.  The dominant allele has a more severe 
phenotype.  Null alleles are essentially wild type.  We have developed 
several lines of evidence to strongly suggest that we have isolated 
rol-6 and that it is a collagen gene.
A cosmid contig (courtesy of A.  Coulson) has been established with 
the left end anchored by zyg-9 and the right end by a cloned Tc1 
element (thanks to J.  Park).  This contig spans a distance greater 
than 300 kb and includes rol-6.  Southern blots of EcoRI digested 
cosmid DNAs probed with the cloned collagen gene, col-2, reveal the 
presence of two collagen genes.  Both genes are on 4 kb EcoRI 
fragments from non-overlapping cosmids (C12G12 and C07B7).  Taking 
advantage of the many deficiencies around rol-6, we have mapped the 
collagen gene in C12G12 to rol-6 using deficiency strains.  Since 
homozygous deficiency chromosomes are lethals, we have selected 
mnDf45/mnDf80 for use in mapping rol-6.  mnDf45/mnDf80 is a viable 
strain in which each homologue of linkage group II carries a slightly 
different, overlapping deletion spanning rol-6.  As a result, rol-6 is 
entirely deleted from these animals.  Hybridization of C12G12 to blots 
of EcoRI digested mnDf45/mnDf80 show that the 4 kb EcoRI fragment 
containing the collagen gene is absent in this genome.  This is the 
expected result if the collagen gene in C12G12 is rol-6.  The collagen 
gene in C07B7 is present in the mnDf45/mnDf80 genome.  The size of the 
overlap between mnDf45 and mnDf80 is between 7 kb and 17 kb.  We are 
making more precise measurements of the size of the overlap.  In 
addition, C12G12 contains one of the ends for mnDf80 and one for 
mnDf85.  Both of these ends are located in an 8 kb SalI fragment 
containing the putative rol-6 gene.  A cosmid (B0382) to the left of 
C12G12 contains one of the ends for mnDf46.  These ends are probably 
the left ends of these deficiencies.  We have not been able to 
identify either end of mnDf45.  Cloning of the dominant allele, su1006,
and the recessive allele, e187, of rol-6 is presently being done.  
Sequencing of the wild type allele is under way.
We have preliminary results from Northern blot analysis using a 2.2 
kb HindIII fragment containing the putative rol-6 gene as a probe to 
egg, L2 to dauer, L3 to L4 and L4 to adult molt total RNAs.  A 1.2 kb 
transcript is seen at the L2 to dauer molt.  A 1.2 kb and a 1.0 kb 
transcript are detected at the L3 to L4 and L4 to adult molts.  No 
transcript is observed in total egg RNA.  These blots were washed 
under high stringency conditions (.03 M Na+, 10% formamide, 65 C).  
The time of expression of the putative rol-6 gene agrees with the time 
of appearance of the rol-6 phenotype.  We cannot rule out the 
possibility of cross hybridization between rol-6 and other collagen 
gene transcripts.  Total L4 to adult RNA from the mnDf45/mnDf80 strain 
has been prepared and Northern blots will be done.  If the transcripts 
are specific for rol-6 then we expect that the 2.2 kb HindIII probe 
will not hybridize to mnDf45/mnDf80 total RNA.
Genomic DNAs have been made from several putative null alleles of 
rol-6 (provided by R.  Horovitz).  Southern blots of HindIII, EcoRI 
and BamHI triple digested genomic DNAs from these strains have been 
made and probed with either the 2.2 kb HindIII or an 8 kb SalI 
fragment containing the putative rol-6 gene and flanking sequences.  
Triple digests were done to facilitate detection of subtle differences 
In the length of restriction fragments associated with rol-6.  Initial 
results have not shown any detectable differences between these null 
strains and N2.  There are still several null genomic DNAs to be 
Attempts at Tc1 tagging rol-6(e187) by crossing e187 into the HH*6 
mutator background and screening for non-rollers have not been 
successful (HH*6 supplied by M.  Finney).  However, a semidominant, 
unlinked suppressor of rol-6(e187) has been isolated from this screen 
and backcrossed six times into N2.  Suppression of rol-6 e187) is 
complete in the homozygous state (e187/e187; sup/sup).  As a 
heterozygote (e187/e187; sup/+), worms roll as L3s but not as adults.  
We have not observed any revertants while maintaining the suppressor 
in the HH*6 background.  Genomic DNAs have been prepared from the 
backcrossed suppressor strain and Tc1 screening is underway.  The 
suppressor is being mapped genetically.
The collagen gene in C07B7 may be vab-9, y 
close to the left of rol-6 and the vab-9 phenotype could be a cuticle 
defect.  We are presently subcloning this collagen gene and beginning 
to do expression studies and molecular mapping.
[See Figure 1]

Figure 1