Tetomerk site position heterogeneity in macronudear DNA of Paramedum primaureha

S In Paramecium p r i m a u r e l i a , the macronudear gene encoding the G surface protein ia located near a telomere. In th is study, mul t ip le copies of t h i s te lomere have been i s o l a t e d and the subte lomer ic and t e l o m e r i c regions of some of them have been sequenced. The te loroer ic sequences consist of tandem repeats of the hexanucleotidea C»A2 or C3A3. We show that the l o c a t i o n where these repeats are added, which we c a l l the te lo roer ic s i t e , i s va r iab le w i t h i n a 0.6-0.8-kb reg ion . These resu l t s are discussed in re la t ion with the formation of macronudear DNA. INTRODUCTION Paramecium, l i ke a l l c i l i a t e d protozoa, exhibi ts a nuclear dimorphism. In the same cytoplasm, coex is t two kinds of nuc le i w i t h two d i f f e r e n t roles : a s i l en t , d ip lo id micronucleua which i s the germinal nucleus and a metabolically act ive, polyploid macronucleus which i s the somatic nucleus (for review, see reference 1). The polyploidy of the raacronucleus is about 800 C for Paramecium (2). The sexual phases of conjugation and autogamy are associated w i th the degradat ion of the o ld roacronucleua and w i t h the formation of a new one af ter d iv is ion of the zygotic nucleus. This last step is known to involve the ampl i f i ca t ion , fragmentation and e l iminat ion of DNA and has been the subject of previous i nves t i ga t i ons i n var ious c i l i a t es (3,4,5,6). Chromosomal DNA fragmentation leads to an increase in the number and a reduct ion i n the s ize of macronudear chromosomes as compared to roicronuclear ones. For instance, Tetrahymena has 5 roicronuclear chromosomes per haploid genome and several hundred macronuclear chromosomes of d i f fe rent sizes ranging from about 2D-kb to 1-Mb (7). Although the exact number of Paramecium roicronuclear chromosomes i s unknown (but appears to be smal le r than 60) see reference 8) , the number and s ize d i s t r i b u t i o n of macronuclear chromosomes analysed by orthogonal f i e l d a l t e r n a t i o n gel e l c t ropho res i s (OFAGE, see reference 9) i s of about the same order aa i n © IR L Pros Limited, Oxford, England. 1 7 1 7 Nucleic Acids Research Tetrahymena. suggesting that f ragmentat ion also takes place during macronuclear development (F. Caron, unpublished results). As a consequence, genes which occupy an in te rna l pos i t i on in micronuclear chromosomes may become teloraeric on raacronuclear chromosomes. A good example i s the rDNA gene of Tet rahymena. The unique micronuclear gene ( integrated copy) and i t s environment have been characterized (10). This gene is amplif ied in the macronucleus to multiple DNA molecules of two kinds which contain either 1 copy (11-kb rDNA) in the developping macronucleus (11) or 2 inverted copies (21-kb rDNA) generated by DNA duplication in the mature macronucleus (12,13). In these molecules, a block of 20 to 70 hexanucleotide C4A2 repeats i s located at each end (14). The te lomer ic region def ined by t h i s block and the subtelomeric regions immediately adjacent to the repeats can therefore be defined without any ambiguity. These aubtelomeric regions are present in the micronuclear regions surrounding the in tegrated rDNA where there are no adjacent c lus te rs of C^A2 (15). In raicronuclear DNA, these subtelomeric macronuclear sequences are adjacent to sequences which are deleted during the process of macronuclear development since they are not found anywhere in the macronuclear DNA (16,17). The analyais of these subtelomeric regions has been developped further. Yao et a l . (17) have determined on the linear integrated rDNA of Tetrahymena the two si tes where breakage occurs to generate chromosomal rDNA, and they have found near both junc t ions a pai r of inverted repeats which supposedly pa r t i c ipa te in the excis ion mechanism. Challoner and Blackburn (18) have aequenced the telomeric and subtelomeric regions of the raacronuclear rDNA molecules of various species of c i l i a t e s in the Tetrahymenina auborder. They have found that the rDNA subtelomeric sequences are conserved for a given species but vary from one species to another. Moreover, w i th the recent discovery of a teloraere te rmina l transfera8e a c t i v i t y in Tetrahymena c e l l f ree ext racts by Greider et a l (19), one can argue that dur ing raacronuclear development, chromosome breakage occurs at spec i f i c s i t es to which C4A2 repeats are added by an enzymatic machinery which does not seem to depend on the subtelomeric macronuclear sequences. I f th is is true, then within a given species, the multiple copies of a given macronuclear telomere w i l l have ident ical subteloraeric sequences and te lomer ic sequences w i l l d i f f e r only by the numbers of repeats. The experiments we describe in t h i s paper on Paramecium pr imaurel ia argue