Worm Breeder's Gazette 7(2): 35

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.

Yeast Segregator Functions in Worm DNA: Putative C. elegans Centromeres

D. Stinchcomb, D. Hirsh

DNA sequences involved in chromosomal replication, segregation and 
structure can be identified by transformation of the yeast, 
Saccharomyces cerevisiae (1-4).  Specific DNA fragments from a wide 
variety of eukaryotes including C.  elegans, permit autonomous 
replication of hybrid plasmids in yeast cells (5).  In addition, the 
termini of the linear extrachromosomal rDNA molecule from Tetrahymena 
are capable of functioning as chromosome ends when introduced into 
yeast by transformation (4).  This conservation of signals responsible 
for chromosome behavior encouraged us to search for C.  elegans DNA 
sequences that would bestow proper segregation behavior upon yeast 
hybrid plasmids.  So far, we have identified and characterized two 
such segregator (SEG) functions from the worm genome.
Segregator functions rescue aberrant segregation of autonomously 
replicating hybrid molecules in yeast cells.  In spite of their high 
copy number and their ability to replicate during each cell division, 
ARS (autonomously replicating sequence) hybrid molecules are not 
propagated efficiently during mitosis.  After 10 generations of growth 
in non-selective media, only 0.1 - 1.0% of the yeast cells retain the 
transformed phenotype.  This mitotic instability can be alleviated by 
the addition of yeast centromeric sequences (2,3).  To search for 
similar segregation functions in the C.  elegans genome, we inserted 
random worm DNA fragments into the yeast vector, YRp17.  YRp17 
contains a putative yeast chromosomal origin of replication (ARS1) and 
a yeast selectable marker: the URA3 gene.  We transformed a ura3 yeast 
strain with the collection of YRp17/worm hybrid plasmids and then 
enriched for mitotically stable Ura+ transformants.
In this fashion, we isolated two hybrid plasmids that are stably 
propagated in yeast.  Both of the original hybrid molecules contained 
one fragment bearing SEG function: SEG1, 7.1 kilobase pairs (kb) and 
SEG2, 3.2 kb.  We then used the Ura+ phenotype of yeast cells carrying 
the YRp17-SEG hybrids to assess mitotic and meiotic behavior of the C. 
elegans SEGs.  In comparison to yeast centromeres, worm SEGs behave 
aberrantly.  After 10 generations of growth in non-selective media, 15 
- 40% of the cells contain the SEG-bearing molecules while hybrid 
molecules with yeast centromeres are present in 40 - 90% of the cells. 
Diploids (ura3 /ura3 
hybrid/O) were induced to undergo meiosis and the four products of 
meiosis were analyzed after tetrad dissection.  If the URA3+ hybrid 
molecule is present in one copy per cell, if it is then replicated 
during premeiotic S phase and subsequently segregated properly, it 
should be found in two of the four meiotic products.  Yeast centromere 
plasmids segregate 2 Ura+:2 Ura- in 57% of the tetrads.  SEG1 and SEG2 
hybrids segregate 2+:2 in 19% and 43% of the tetrads, respectively.  
In contrast, YRp17 alone produces no 2+:2 tetrads.  In 98% of the 
yeast centromere 2+:2- , the sister chromatids do not separate until 
the equational, second meiotic division.  Only 65% and 35% of the SEG1 
and SEG2 2+:2- tetrads show normal second division disjunction; the 
remainder separate precociously.
SEG1 and SEG2 are both linked to sequences that are repeated in the 
C.  elegans genome.  SEG1 hybridizes weakly to 6 or 7 EcoRI fragments 
in addition to the homologous 7.1 kb fragment.  SEG2 hybridizes to 
some 30 other bands.  The two patterns of hybridization are different; 
likewise, SEG1 and SEG2 do not cross-hybridize.  Neither SEG1 nor SEG2 
hybridizes to yeast centromere sequences.  If the repeated sequence is 
associated with SEG function (and we have yet to demonstrate a strict 
correlation) then SEG1 and SEG2 may be members of two different 
repeated classes of worm segregators.
SEG1 and SEG2 represent putative C.  elegans centromeres.  They were 
found at reasonably high frequency: one every 500 - 2000 kb (a very 
rough estimate, replete with error-prone assumptions).  Such a high 
frequency is consistent with the observation that C.  elegans 
chromosomes are polycentric.  However, other functions for segregators 
are imaginable (e.g.  nuclear membrane or matrix binding).  
Furthermore, SEG1 and SEG2 may not behave as segregators in C.  
elegans.  Microinjection will provide the means for assaying their 
segregation in worm cells.  If the SEG sequences allow hybrid 
molecules to segregate properly, they may be ideal vectors for the DNA 
transformation of C.  elegans.