Worm Breeder's Gazette 8(1): 40

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.

An Approach to Cloning C. elegans Genes Defined Only by Mutations: Identification and Mapping of Closely linked Tc1 Dimorphisms

G. Ruvkun, V. Ambros, M. Finney, J. Park, B. Horvitz

The genetic characterization of certain genes involved in C.  
elegans development and behavior has proceeded to the point that an 
important next step is the determination of the molecular structures 
of these genes.  For example, we wish to clone DNA segments that 
contain the genes lin-14, unc-105, 
nventional approaches such as cDNA cloning 
are not appropriate for these genes, which have been identified only 
by mutations; we have no evidence as to the identity or even the 
existence of protein products from these genes.  Rather the approach 
we are taking is to identify DNA restriction fragment length 
polymorphisms (RFLPs) that genetically map adjacent to these genes and,
by isolating genomic clones that contain the particular RFLP and 
adjacent regions, 'walk' towards the desired gene.
Our experimental strategy to find a RFLP closely linked to each of 
the genes listed above begins with the observation that C.  elegans 
strain Bergerac (RW7000) has approximately ten times more copies of 
the mobile genetic element Tc1 than strain Bristol (Emmons et al.  
Cell 32, 55, 1983); our data indicate that Bristol contains 29 Tc1 
elements.  If one assumes that the approximately 300 Tc1 elements in 
Bergerac are randomly distributed in the genome and compares this 
number to the total of about 300 map units in the C.  elegans genetic 
map, then the expectation is that there will be one Tc1 in strain 
Bergerac per map unit.  Nearly all of the Bergerac Tc1s must be 
missing in Bristol, so that the two strains are dimorphic at these 
loci.  These Tc1 dimorphisms can be mapped relative to visible genetic 
markers to yield a readily isolated set of physical probes with known 
genetic locations for walking in the C.  elegans genome.
Our approach has been to genetically map all Tc1 elements in strain 
Bergerac around the genes lin-14 X, unc-86 III, unc-105 II, and rol-6 
II and to clone restriction fragments that contain those Tc1 elements 
mapping closest to each gene.  We discuss below one protocol we have 
used for mapping and cloning the Tc1 elements around lin-14.  Similar 
protocols have been used for unc-86, noIn 
principle, Bergerac Tc1 elements can be located on the Bristol genetic 
map by treating each Bergerac Tc1 element as an unselected marker in a 
three-factor cross with a doubly marked Bristol chromosome, in a 
manner similar to that used to map a specific Tc1 RFLP near the actin 
gene (Files et al., 1983, JMB in press).  The presence or absence of 
Bergerac Tc1 elements is then assayed by Southern blot analysis of DNA 
prepared from a number of recombinant strains, using Tc1 as a probe.
In practice, the detection of the linkage behavior of Tc1 elements 
in F2 Bristol/Bergerac recombinant strains is nearly impossible 
because such a strain can have up to 300 other Tc1 elements unlinked 
to the region of interest.  To decrease the number of extraneous Tc1 
bands in Southern blot analyses, we have repeatedly backcrossed hybrid 
animals to Bristol while selecting for the Bergerac allele of the 
desired gene.  Backcrossing may be done before, after, or during the 
selection of recombinants; the number of backcrosses should be at 
least eight.
In the case of lin-14, the wild-type Bergerac lin-14 allele ('lin-14(
Berg+)n) was selected for 10 cycles of backcrossing to a recessive 
Bristol lin-14(n179) allele in the following way: in the first cycle 
lin-14(n179) males were mated with lin-14(Ber hermaphrodites to yield 
lin-14(n179)/lin-14(Berg+) hermaphrodites.  These were mated with lin-
14(n179) males and from that cross wild-type lin-14(n179)/lin-14(Berg+)
hermaphrodites were used for the next cycles of backcrossing with lin-
14(n179) males, and so on.  (In all matings probable cross-progeny 
hermaphrodites were picked based on the presence of a high percentage 
of cross-progeny males on the same plate.) After the final cross, 
homozygous lin-14(Berg+) hermaphrodites were isolated for DNA 
isolation and subsequent crosses.  Such a protocol should remove all 
Bergerac Tc1 elements on chromosomes other than X and preserve only 
those Tc1 elements within about 10 map units on either side of lin-14.  
DNA was prepared from a culture of the lin-14(Berg+) strain congenic 
to Bristol, and this DNA was probed with Tc1 in a Southern blot and 
compared to the Bristol and Bergerac EcoRI patterns of Tc1 
hybridization bands.  We found 20 additional Tc1 hybridization bands 
in this strain, which we hoped were those Tc1 elements on the g 
chromosome near lin-14.To order these 20 Tc1 elements relative to each 
other and relative to lin-14, we performed two 22-factor crosses.  The 
X chromosome from the congenic strain was mated into two different 
doubly marked Bristol strains: dpy-6 
sma-5, X-linked markers separated by 2.
0 and 0.5 map units, respectively.  In the first case, 20 Lin non-Dpy 
recombinant animals and in the second 8 Lin non-Sma animals were 
picked in the F2, and homozygous strains not segregating Dpy or Sma 
animals were isolated.  DNA was prepared from cultures of each of 
these strains and probed with Tc1 in a Southern blot analysis to 
determine the presence of each of the 20 extra Tc1 bands of the 
congenic strain.  Because we were selecting for Bristol lin-14 in the 
recombinant animals, those Tc1 bands closest to lin-14 in Bergerac 
would not be found in the Lin non-Dpy and Lin non-Sma animals picked.  
Three Tc1 elements behaved in this way and therefore mapped very close 
to lin-14.  Two Tc1 hybridization bands found in some of the Lin non-
Dpy recombinants but none of the Lin non-Sma recombinants were 
assigned to between dpy-6 and lin-14.  Similarly one Tc1 mapped 
between lin-14 and sma-5.  In addition, many of the Tc1 hybridization 
bands outside of the dpy-6  could be ordered 
relative to each other and roughly mapped by their order of 
disappearance in the recombinants due to unselected second 
recombination events outside the selected interval.
The total number of Tc1 elements we have localized to this 2.5 map 
unit interval around lin-14 is six or about twice as many as expected. 
In addition three Tc1 elements are very closely linked to lin-14, 
apparently 0.1 to 0.2 map unit or less from lin-14.  We have similarly 
located multiple closely linked Tc1 elements around unc-86 (seven Tc1 
elements within 0.1 map unit, four of which have not yet been 
separated from Bergerac unc-86) and unc-105 (two Tc1 elements within 0.
3 map unit).  It is possible that these apparent Tc1 clusters reflect 
recombinational suppression similar to that which might be the cause 
of the clustering of visible markers on C.  elegans chromosomes.
We have begun our walk around lin-14 by cloning two of the Tc1 
elements near lin-14, using cloned Tc1 as a probe to screen a genomic 
library constructed from size-fractionated DNA isolated from the 
congenic lin-14(Berg+) strain.