Worm Breeder's Gazette 12(3): 93 (June 15, 1992)
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.
Two TR679 -derived unc-33 mutations, mn260 and rh1 O30(the latter given to us by Ed Hedgecock), are insertions of Tc4 elements at identical sites within the unc-33 coding sequence. The mn260- Tc4 element is 1.6 kb in size and appears to be very similar to the Tc4 elements in ced-4 and unc-86 described by Yuan et al. (1991, Proc. Natl. Acad. Sci. USA 88: 3334-3338), who showed that the 1.6-kb elements contain almost perfect inverted terminal repeats of 774 base pairs. Because the rh1O30- Tc4 element was 3.5 kb in size, we cloned and sequenced it. We found a 2342-bp sequence in rh1O30- Tc4 that was not present in the previously-studied Tc4 elements; it replaced a 477-bp segment in the middle of one of the inverted repeats of the l.6kb Tc4 element [See Figure 1]. When a 2-kb fragment from within the novel 2342-bp sequence was used as a probe on a Southern blot with N2 and TR679 genomic DNA digested with enzymes that do not cut any known Tc4 elements, 4-5 bands in each strain were identified. N2 and TR679 have about 25 and 30 total Tc4 -hybridizingbands, respectively; the 4-5 bands recognized by the 2-kb probe were a subset of these Tc4 -hybridizingbands.
On northern blots with poly(A)-enriched RNAs prepared from N2 and TR679 ,the 2-kb probe did not detect any message, even though the 1.6-kb Tc4 element hybridized to a number of bands on northern blots with N2 and TR679 RNA. We also used the 2-kb probe to screen two different N2 cDNA libraries (provided by J. Ahringer and J. Kimble and by R. Barstead and R. Waterston). Three cDNA clones were isolated and together they represent 2.1 kb of a message, including a poly(A) tail. The 5' end of the cDNA sequence lies within the Tc4 inverted repeat and extends through most of the novel 2342-bp sequence (see figure). We have not mapped the 5' end(s) of the transcript(s) that the cDNA clones represent. The poly(A) tail begins 11-12 nucleotides downstream from a potential poly(A) signal AATAAA. The 2342-bp cDNA sequence contains five exons (all four introns are very short) within the 3.5-kb Tc4 element; an open reading frame with a coding capacity for 564 amino acids is entirely within the novel 2342-bp sequence. The putative Tc4 protein is very hydrophilic and positively charged with a pI of 8.83. It does not have high similarity to any other polypeptide in the databanks.