Worm Breeder's Gazette 13(1): 32 (October 1, 1993)
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.
We have employed the transposable element Tc1 as a polymorphic marker in order to define the evolutionary relationships between strains of C elegans. We first determined the number of Tc1 elements in strains N2 , PA2 , C12 aand G a1 ,reported to be low-copy number strains and RC301 , TR403 , DH424 , RW7000 and BO, described as high-copy variants (1). N2 is commonly referred to as having 30 Tc1s in its genome, while the values reported for BO vary between 300 and 500. No specific values have been reported for the other strains mentioned above.
We utilized two different approaches to determine the Tc1 copy numbers. Initially, genomic DNAs from each strain were digested with EcoRI and separated on large, slow-running 0.8% agarose gels (23cm, 20V, 96 hours). Southern blots of these gels were then probed with a 1.6 kb cloned Tc1 element (2). Tc1 elements of the low-copy number strains could be resolved sufficiently well on these blots to allow direct enumeration from the autoradiographs. The results obtained from these blots for the low-copy strains are shown in Table 1. Tc1 copy numbers varied between 26 and 30 and the overall patterns were similar with minor differences between strains. RC301 ,contrary to previous reports (1), appeared to be a low-copy strain, containing 26 Tc1 bands. Re-analysis of fresh RC301 stocks, re-ordered from the Caenorhabditis Genetics Center and kindly provided by J. Hodgkin (MRC, Cambridge, England), confirmed our initial results. The band patterns of GA1 and N2 were identical. Other low-copy strains were distinct from N2 and from each other, in their Tc1 band patterns, but retained 21-22 bands with mobilities indistinguishable from N2 bands. C12 aand PA2 were clearly different from N2 but were nearly identical to each other differing at only one band. RC301 appeared to be the most distant relative of N2 based on the number and pattern of its Tc1 elements.
DNA dot-blots were utilized to analyze the Tc1 copy numbers of the high-copy strains. Genomic DNAs from N2 , RC301 , TR403 , DH424 , RW7000 and BO were blotted at four different dilutions and were probed with the cloned Tc1 element. The values for DNA loads were corrected by probing the same blot with a cloned single-copy gene fragment (2). The blots were analyzed by direct phosphor-imaging of ß-emissions on a Phosphor Imager Computing Densitometer (Molecular Dynamics). Intensities of phosphor-images were integrated directly using ImageQuant software and the results are summarized in Figure 1. All values were within the linear range of the phosphor- screen extending over a 105 range of signal intensities thus ensuring reliable quantitation. Using RC301 as a reference standard, at 26 Tc1s ,the Tc1 copy numbers for the strains TR403 , DH424 , RW7000 and BO were estimated to be 205, 260, 410 and 494 respectively, based on least-squares linear regression. The slope for RW7000 ,a separately maintained stock of Bergerac-BO, has a large standard error and is not significantly different from that of BO. Nevertheless, the difference could be genuine, since different levels of Tc1 -transpositionmay have accumulated in these two stocks of an active mutator strain. In all other cases, the lines fit the data well and thus the values determined should be highly accurate.
References:
(1) Cassada, R. WBG 9(3):29, 1986; Hodgkin, J. WBG 10(2):140-141, 1988; Hodgkin, J. WBG 11(5):60, 1991.
(2) Egilmez, N.K. & R.J. Shmookler Reis, manuscript in preparation.
