Worm Breeder's Gazette 11(2): 78
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.
In order to understand the mechanisms underlying Tc1 excision and transposition, we are studying transcription and translation of the Tc1 open reading frame. To identify the 5' end of a possible Tc1 message, we have performed primer extension experiments with polyA+ RNA from various strains as the template. Using three primers located at various sites within the Tc1 ORF, we have identified a number of potential 5' ends. These 5' ends result in primer extension products that are distinct from the products of premature termination of reverse transcriptase seen when sense RNA synthesized in vitro is used as the template. To gain further evidence for transcripts initiating within Tc1, we carried out PCR amplification experiments after first strand cDNA synthesis from polyA+ RNA preparations. We wanted to know whether there was a region of Tc1 that could not be amplified in this way, suggesting the presence of a 5' end. So far the longest region we have amplified spans from nucleotide 304 to 1548 (putative polyadenylation signal). Our inability so far to amplify with two different upstream primers in the region adjacent to the inverted repeat (nucleotides 60 through 100), and a downstream primer at position 924-943, suggests that the 5' end may be located in the region between nucleotide 60 and nucleotide 304, and that no read through transcripts extend past position 943. As reported at the Cold Spring Harbor meeting in May, we have three antisera that recognize TcA. Two were raised against synthetic peptides, and one against a TrpE-TcA fusion protein. After purification by binding to antigen, these antisera reacted primarily with a single protein of 34-36 kd in nematode homogenates from various strains. We have now found that this protein has a higher molecular weight than the 273 aa TcA protein synthesized in E. coli (kindly provided by R. Plasterk). This result is similar to the one reported by R. Plasterk at the meeting. Therefore, the protein in nematodes either has additional amino acids, or is otherwise modified, or it is an unrelated, crossreacting protein.