1. .
Conclusions
All breakage site locations examined were conserved, i.e. no variation in
the location of Cbs-s was observed among the species examined. Thus,
there is evolutionary pressure for retaining chromosome breakage sites at
the locations studied.
At a given chromosome breakage site, more than one variant of the Cbs
sequence can occur in related species.
Tetrahymena thermophila, a ciliated protozoan, is a unicellular model organism with two
different nuclei: a diploid ‘germline’ micronucleus (MIC), and a ‘somatic’ polyploid
macronucleus (MAC). A new macronucleus develops during conjugation from the zygotic
micronucleus. This development includes fragmentation of chromosomes into about 200
subchromosomal pieces. The object of this study is to use comparative genomics to
determine the importance of the locations and sequence features of fragmentation sites.
All fragmentation sites have been determined in the reference species, T. thermophila;
these positions, containing a 15-basepair chromosome breakage signal, can also be
deduced from analysis of the macro– and micronuclear DNA databases. Corresponding -
or non-corresponding – fragmentation sites in other species can be identified with some
degree of certainty from the macronuclear databases of other species and then isolated
experimentally using the polymerase chain reaction.
We studied two locations where chromosome fragmentation occurs in the reference
species. The analysis of homologous regions in three related species has been completed
at one of these locations. We found that two of the species had the same ‘classical’ 15
base pair chromosome breakage signal as found in the reference species, whereas in the
third species it differed by one nucleotide.
At a second site, where chromosome breakage occurs in the reference species, one of its
relatives has a longer, unbroken chromosome according to the database. We determined
that this relative has a chromosome breakage signal at the homologous location, indicating
faulty assembly in the database. Thus, the locations of chromosome breakage sites were
conserved in all cases.
Programmed chromosome fragmentation in Tetrahymena.
A comparative genomic study.
Courtney Taylor*, Kylie Corry*, Logan Roberts*, Benjamin Clevenger and P. Huvos (faculty mentor) *The first three contributed equally
Department of Biochemistry and Molecular Biology
Tetrahymena thermophila has two different kinds of nuclei: a diploid
micronucleus (MIC), and a polyploid macronucleus (MAC).
The ‘germline’ micronucleus has 5 pairs of chromosomes.
It is transcriptionally inactive during the vegetative phase of
the life cycle, but takes part in the sexual processes that
occur during conjugation.
The 'somatic' macronucleus contains many copies of
fragmented chromosomes. It is transcriptionally active and
is responsible for directing most functions in the cell.
Orias 20000
ABSTRACT
A new macronucleus develops after each conjugation from the zygotic
micronucleus. The micronucleus undergoes two types of programmed genome
rearrangement during this development.
(a) Deletion of Internal Eliminated
sequences (IESs) followed by re-
ligation of DNA.
(b) Deletion of Breakage Eliminated
Sequences (BES) associated with
breakage of chromosomes into
smaller DNA pieces.
Inside the BES there is a 15 bp
Chromosome Breakage Sequence
(Cbs).
The Cbs sequence is TAAACCAACCTCTTT/ AAAGAGGTTGGTTTA at
about half of the ~200 chromosome breakage sites. At the other sites,
variants of this sequence are found with one or two nucleotide changes,
mainly at the ends of the sequence (Hamilton et al. 2006).
AIM OF THE STUDY
To gain insight into the chromosome breakage events by examining
the significance of the locations and sequences of Cbs sequences by
comparison with closely related species.
Specifically
v To seek versions of the breakage signal at homologous locations,
and to characterize structural features of the surrounding sequences.
v To analyze locations where breakage signals are generated or lost
to identify the mechanism of such changes.
Mochizuki and Gorovsky 2004
Searching for Chromosome Breakage Sites in relatives of
the reference species, T. Thermophila in the area of the fourth
chromosome breakage site (Cbs 4) of the MIC chromosome
studied (‘supercontig 6’ )
Examining Supercontig 46 of T. elliotti, which has been
assembled in the database as a contiguous chromosome
spanning the region of Cbs 1.
In the area of Cbs 4, we were able to amplify the sequences intervening
between neighboring macronuclear (MAC) chromosomes. The PCR products
were purified following gel electrophoresis and then sequenced.
The sequences of the products (prod) were aligned with a composite (comp)
made from the ends of the neighboring MAC chromosomes at Cbs 4.
The missing sequence in the composite corresponds to the region
excised from the developing macronuclear DNA.
Fragments eliminated at the breakage sites:
STRATEGY AND METHODS
Micro- and macronuclear databases are available for the reference species, T.
thermophila, and macronuclear databases for three relatives (T. malaccensis,
T. elliotti, and T. borealis).
We examined a 2.5 kb long contiguous micronuclear sequence of the
reference species that contains five Cbs-s and is broken into six MAC
chromosomes. The corresponding regions in the other species were
identified by sequence alignment. Polymerase chain reactions (PCR) from
the ends of neighboring MACronuclear sub-chromosomes were used to
amplify the intervening sequence in the MICronucleus between the two sub-
chromosomes. The products were sequenced, and the sequences compared
by alignments (BLAST, LALIGN, NEEDLE).
In the area of the first chromosome breakage site (Cbs 1), the database showed
that the sequence was contiguous in one of the related species (T. elliotti).
This would mean that in T. elliotti there is no functional Cbs at that location.
When we aligned this sequence (called TelS46database) with the MIC
sequence of T. thermophila (Ttsup6-Cbs1), the section in the vicinity of the
Cbs was represented by ‘Ns’ in the T. elliotti sequence, showing that it could
not be sequenced.
We used the flanking sequences for the design of primers ‘forward’ and
‘reverse’ to amplify this region. We were able to sequence the entire
product, including the region represented by Ns in the database, and
identified a Cbs-like sequence (AAAACCAACCTCTTT) in it that is very
likely to be functional.
Consequently, the PCR product must be derived from the MICronucleus.
Also, the sequences to the left and right of this region should be on separate
MACronuclear chromosomes, and be in the vicinity of chromosome ends, i.e.
telomeres. To test for this, the primers that had been used to amplify the un-
sequenced region, were used in combination with telomer primers.
Amplification products with molecular weights that are consistent with the
distance of the primers from telomers were obtained. This confirms that the
Cbs identified in the T. elliotti sequence is functional. Thus, the sequence in
the database was assembled erroneously.
3000 bp
2000 bp
1000 bp
800 bp
500 bp
400 bp
300 bp
200 bp
600 bp
Agarose gel electrophoresis of PCR products
obtained with the following
primer combinations:
A
B C
A
‘forward’ and telomere primer
B
‘reverse’ and telomere primer
C
‘forward’ and ‘reverse’
*The macronuclear sequence to the ‘right’ of the Cbs is missing from the
database