PROGRESS IN PROTOZOOLOGY

256 D. L. NANNEY
23-33%. Later workers (S u y a m a 1966, Allen and Gibson 1971,
Flavell and Jones 1970), reported values different from Sueoka's
for some particular strains, in some cases because of the use of
different techniques, in some cases because of the use of strains of
questionable provenance (Borden et al. 1973b). But all workers,
regardless of the techniques used, found large strain differences in the
DNA composition.
The variation in the G + C°/o in T. pyriformis strains is over twice
that found in the vertebrates. If, to take an utterly simplistic view, the
base-ratio variation in a group of organisms were directly proportional
to the time since they had a common ancestor, we could use the vertebrates,
whose age we know, to calibrate the instrument and measure the
age of the T. pyriformis complex. The vertebrates with 4°/o variation
have a common ancestor about 500 million years ago; an 8°/o variation
in Tetrahymena would signify a common ancestor over 1000 million
years ago, over halfway back to the origin of the eukaryotes.
The G + C ratio, however, is a crude measure of genetic relationships,
and susceptible to unknown perturbances. One needs a more refined
measure of molecular similarities. Such a measure, though still
with its complications, is provided by DNA-DNA hybridizations. In this
procedure the double-standed DNA is "melted" and then allowed to
reanneal with DNA from another source. If a foreign DNA is identical
to that used as the reference, cross^annealing should be equivalent to
reannealing. As differences accumulate, affinities decline and the fraction
of reannealing gradually drops. The interpretation of nucleic hybridization
data requires some brief discussion. We need to note, for example,
that the conditions of reannealing influence the specificity of the
results. Moreover repetitive and unique sequences have different hybridization
kinetics, and they may have different evolutionary stabilities.
The main comparative hybridization study of total, cellular (largely
macronuclear) DNA in Tetrahymena is that of Allen and Li (1974).
They used T. thermophila as the reference species and prepared DNA
from the other mating species available at the time (Table 8). They
separated the repeated from the unique sequences, and these are separately
considered. The unique sequences were hybridized first under
relatively non-stringent conditions, at 50°C. Obviously, none of the
species is close to T. thermophila, but they show a gradation; T. pigmentosa
is relatively closer than the others, even though it shows only
about 30% reannealing. This percentage drops sharply when hybridization
is carried out at 65°C, when more complete complementarities are
required, and the only approximately identical structures fall
apart. All the species studied show only about 5% reannealing at 65°.
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