Copyright 0 1991 by the Genetics Society of America
Permanent Rescue of a Non-Mendelian Mutationof Paramecium by
Microinjection of Specific DNA Sequences
Helen Jessop-Murray, Linda D. Martin, DavidGilley, John R. Preer, Jr.,and Barry Polisky
Program in Molecular, Cellular, and Developmental Biology, Department of Biology, Indiana University,
Bloomington, Indiana 47405
Manuscript receivedMay 10, 1991
Accepted for publication August 2, 1991
ABSTRACT
The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its
surface. Although the A gene is intact in the micronuclei of d48, the A gene copies inthe macronucleus
contain a large deletion eliminating virtuallythe entire coding sequence. Previous studies
showed that
microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently
restored A expression after autogamy. Together with other data,this result suggeststhat in wild type
cells the A gene in the old macronucleusensures the presence of a cytoplasmic factor that prevents A
gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old
macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular
mechanisms involved in this unusual
phenomenon, we attempted to define the region(s) of the A gene
necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is
sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued
strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments
of the A gene have the ability to rescue d48 to different extents, some being more efficient than
others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might
act as or encode a cytoplasmic
factor. Molecular mechanisms that may be involved in the rescue of
. d48 arediscussed.
I
N wild-type Paramecium tetraurelia stock 51, the A
surface protein is coded by the A gene located
surfaceantigengene
is aberrant. A largechromosomal deletion which begins near the 5’ end of the
gene (EPSTEINand FORNEY 1984; FORNEYBLACKand
close to the telomere in macronuclear chromosomes
BURN 1988) eliminates virtually the whole
A gene from
(reviewed by PREER1986). In each wild type cell, the
the macronucleus.
polyploid macronuclear chromosomal DNA contains
T h e genetic behavior of the d48 mutation
is unabout 2000 copies of theA gene. Macronuclear chrousual. A cross of 5 1 (A+)to d48(A-) usually yields A+
mosomes range in size from about 100-600 kb. The
micronucleus (containing about2 100 kbper average
exconjugant F1 clones from the A+ parent, and Achromosome; PREER1986) is diploid. T h e A gene is
exconjugant clones from the A- parent, despite the
present in the micronucleus of a mutant line called
fact that both exconjugants are genetically identical.
d48; however the macronucleus of this mutant conProduction of anF2 by the induction of autogamy in
tains few, if any copies of the intact
A gene (RUDMAN the F1 usually yields no further change in theability
et al.1991). As a result, theA protein is not detectably
to produce the A serotype in the progeny of either
expressed by d 4 8 (EPSTEINand FORNEY1984). At
exconjugant (EPSTEINand FORNEY 1984;RUDMANet
autogamy (a self-fertilization process that occurs pea l . 1991). Since one-half of the F2 exconjugants deriodicallyin P. tetraurelia) and conjugation, anew
scended from the d48 parent are
homozygous for the
macronucleus and new micronuclei are formed from
wild type A gene, the inability to observe proper A
DNA processingofthe
old micronuclei. T h e old
gene processing implies the existence of factors in an
macronucleus degenerates as the new macronucleus
A+ cell responsible for proper processing of the A
develops. Although the molecular details of autogamy gene. Thisnon-Mendelianpatternofinheritance
and conjugation are unknown in Paramecium, the
shows that the genetic difference between d48 and
DNA processing steps involved in generating
polywild type does not lie in the micronuclei of the two
ploid macronuclearchromosomesfromdiploid
mistrains.
cronuclei include DNA cleavage, telomere addition
However, the genetic behavior does not seem to
and DNA amplification. During formation of the ma- represent true cytoplasmic inheritance either. HARUcronucleus in the d48 mutant, processing of the
A
MOTO (1986) showed that transfer of macronuclear
Genetics 1 2 9 727-734 (November, 1991)
728
H. Jessop-Murray et al.
material, but not cytoplasm, from vegetative
wild type
cells into d48 will "rescue" d48, permanently restoring
its ability to produceA after autogamy. KOIZUMIa n d
KOBAYASHI
(1 989) found that microinjection ofwildtype cytoplasm rescued d48, but only if both donor
and recipient were undergoing autogamy. Rescued
cells continued to express A evenafter successive
autogamies. We interpret these results to mean that
the basis for the d48 mutation is not due to the lack
of cytoplasmic hereditary determinants, but instead
results from the absence of a cytoplasmic factor that
is normally produced during autogamy and conjugation in wild type cells. Its genetic determinant resides
within the old macronucleus and is necessary for inclusion of the A gene into thenewly forming macronucleus.
Furtherlight was shedonthisphenomenon
by
microinjection of plasmid DNA containing the
A gene
(GODISKAet al. 1987). When such DNA is injected
into the macronucleus of the A- mutant d12, which
lacks the A gene in its macronucleus, the plasmid
is
linearized, acquires Paramecium-type telomeres, and
(GILLEY
replicates autonomously in the macronucleus
et al. 1988). The injected d l 2 cells acquire the ability
to transform to A+, but "revert" at autogamy when
the old macronucleus is replaced by a new one (GODISKA et al. 1987). T h e elimination of the macronucleus
at autogamy results in the
physical loss of the injected
DNA. These results suggest that the d l 2 mutation
involves an alterationof the micronuclearcopy of the
A gene.Similartransformationtoan
A+ serotype
occurs when the plasmid DNA is injected into the
a n d KOBAYASHI
1989),
macronucleus of d48 (KOIZUMI
exceptthat inthiscase
the cells remain A+ after
autogamy, i e . , d48 is permanently rescued. YOU et al.
(1991) have recently shown that a fragment of the A
gene is capable of d48 rescue. These results suggest
that the d48 phenotype is due tothe deficiency of the
A gene or sub-A gene sequences in the old macronucleus. Presumably, in wild type cells these sequences
a
in the old macronucleus ensure the presence of
cytoplasmic factor at autogamy and conjugation that
prevents loss of the A geneinthe
newly forming
macronucleus. This factor is not present in the cytoplasm of vegetative cells. Once theA gene is lost from
the macronucleus in the mutant d48,
is not
it normally
restored at either conjugationor autogamy.
T h e specific molecular mechanisms involved in this
unusual phenomenon are of interest. To elucidate
thesemechanisms we are attempting to define the
region(s) of the A gene necessary for rescuing d48.
W e show that injection of a 4.5-kb internal fragment
of the A gene yields proper processing in d48 a n d
leads to permanent rescue to wild
the type phenotype.
A
Neither detectable A gene expression nor complete
gene mRNA is required for rescue. We also show that
various fragments have the ability to rescue d48 t o
different extents, some being more efficient than others.
MATERIALS AND METHODS
Strains and culture conditions: Wild-type cells were P.
tetraurelia, stock 5 1. Strain d48 was obtained from stock 5 1
by X-ray mutagenesis and antiserum selection (EPSTEIN
and
FORNEY
1984). Cells were cultured in 0.15% Cerophyl (Pine
Brothers, Kansas City, Missouri) supplemented with 0.1 g/
liter Bacto Yeast extract, 1 mg/liter stigmasterol, 0.45 g/
liter NanHP04,and inoculated with Klebsiella pneumoniae
24-48 hr before use.
For microinjection, autogamy was induced in d48 by
starvation. d48 cells were allowed to undergo six fissions at
27" post-autogamy. Samples were also culturedat 34",
which induces serotype A expression in stock 5 1 (wild type),
and tested with antiserum to confirm that cells had not
spontaneously revertedto wild type. After injection of
cloned DNA, each injected cell was isolated into adepression
slide and cultured for 24 hr at 34", and then a further 24
hr at27 " (about eight fissions). Atthis point, a 20O-pl sample
from each depression was stored at 14" as a preautogarny
stock. The remaining cells were screened for the presence
of cloned DNA by either: testing for serotype A expression,
or by transferringto 20 mlof medium in a test tube,
culturing for 24-48 hr at 27", and analyzing the cellular
DNA by dot-blot hybridization (see below). Following
either
procedure, the lines were then subcultured in tubes at 27"
for 22-26 fissions past autogamy. Autogamy was induced
by starvation and confirmed by staining. Each line was
subcultured for four fissions at 27" postautogamy. To induce serotype expression, fresh medium was added daily to
double the culture volume for 4 days at 34" (EPSTEIN
and
FORNEY 1984), after
which lines were scored for antigen A
expression.
Microinjection: Microinjection of cloned DNA into the
macronuclei of d48 was done essentially as previously deet al. 1987). One difference was that
scribed (GODISKA
plasmids pSA5.5, pSA2.8, pSA4.5, pSA3.O and pSA2.5 (see
below) were injected as cleared lysate preparations (MANIATIS, FRITSCH
and SAMBROOK
1982). The cloned DNA was
dissolved in DRM medium (1 14 mM KC1/20 mM NaC1/3
mM NaH2P04, pH 7.4) at 1 mg/ml.
Cloned DNA: Plasmid pSA 14SB consists of
a 14-kb insert
(sequences- 1590 to 1 1825)
containing the antigen A gene
and aportion of the flanking regions, cloned into the vector
pT7/T3-18 (Bethesda Research Laboratories). The sequencing number system used here refers to the A nucleotide of the ATG codon thought to be the first codon of the
A gene as + l . Plasmid pSA12.8 (containing sequences -264
to +11825) was constructed from pSA14SB by cleavage at
position -1590 with Sal1 and treatment with exonuclease
III/mung bean nuclease (MANIATIS,FRITSCH
and SAMBROOK
1982). The Klenow fragment of DNA polymerase I was
used to fill in the sticky ends and blunt end ligation was
carried out according to MANIATIS,FRITSCH
and SAMBROOK
(1982). Plasmid pSA5.5 (containing sequences +13 to
+5482) was made by digesting pSA14SBwith XmnI and
Ssp1 and isolating the 5.5-kb fragment. BamHI linkers were
attached to the blunt ends (MANIATIS, FRITSCHand SAMBROOK 1982) and theinsert was ligated into the BamHI site
ofpT7/T3-18.
PlasmidpSA2.8
(containing sequences
+403 to +3175) was made in an identical manner to pSA5.5,
except that the insert was a 2.8 kb HgaI fragment whose
sticky ends were filled in withKlenow fragment (MANIATIS,
+
Rescue of a non-Mendelian Mutation
-
-1690
pSAl4SB
+
-284
pSA12.8
.
P
1¶
pSA6.6
¶176
pSA2.8
6482
1
1
Xmrl
Srpl
pSAZ.6
+
401
4617
I
1
Xmrl
I¶
pSA3.0
+
w77%
I 3
pSA4.6
I
JdZ==@l
PSI1
2971
I
+
-I+
+I-
FIGURE1 .-Map of the cloned A gene and subcloned fragments.
The light bar of pSA14SB represents the coding region of the
gene, and the darkbars the flanking regions. pSA5.5,pSA2.8,
pSA4.5,pSA3.0 and pSA2.5 wereconstructedfromfragments
located within the A gene coding region as describedin MATERIALS
AND METHODS. The ability of the fragments to rescue d48 by
microinjectionare indicated by + (rescue),- (no rescue), -/+ (poor
rescue) and +/- (intermediate rescue). Stock51macronuclear
DNA contains the complete A gene, but d48 macronuclearA genecontaining chromosomes lack downstream sequences beyond the
vicinity of + 1 , thereby rendering d48 unable to express the A
surface antigen.
FRITSCHand SAMBROOK
1982) before attachment to the
linkers. PlasmidpSA4.5
(containing sequences +13to
+4517) was an XmnI-Pstl fragment of the A gene ligated
into the SmaI-PstIsitesof pT7/T3-18. Plasmid pSA3.O
(containing sequences + I 3 to +2971)was constructed from
an Xmnl-EglII fragment ligated into the filled-in HindIIIBglII sites of pT7/T3-18. Plasmid pSA2.5 (containing sequences +2971 to +5482) used the same vector sitesas
pSA3.0, except that the A gene fragment was a BglII-Ssp1
digest. Maps of all these plasmids are shown in Figure 1.
Screening preautogamy injected d48 cells for cloned
DNA: Screening was performed on preautogamy injected
lines that had been transferred from depressions to 20 ml
of medium in test tubes and subcultured to 1000 animals/
ml (cells were 16-20 fissions past the previous autogamy).
Either of two methods was used to extract and analyze
DNA: (1) 19,000 animals (3 pg DNA) were resuspended in
0.02 ml of their own culture fluid and addedquickly to 0.04
ml NDS medium (1% SDS/O.5 M Na2-EDTA/lO mM TrisHCI, pH 9.5) at 65" for 24-48 hr. Lysates could be stored
at 4". One microliter of 3 M NaOH was added to 10 pl of
the NDS mixture, and after heating to 65" for 30 min to
denature the DNA, samples were rapidly cooled on ice and
neutralized with 11 pl of 2 M NH40Ac. Duplicate 1-pl
samples (20 ng DNA) were spotted onto a pieceof dry
nitrocellulose. (2) Fifty microliters of 3 M NaOH were mixed
with 500 PI (500 animals/75 ng DNA) of each culture and
heated to 65" for 30 min. After cooling on ice and neutralizingwith 37.5 p1 of 7.5 M NH40Ac, each sample was
transferred to nitrocellulose using a Schleicher and Schuell
minifold I system.
Paramecium DNA preparation:NDS lysates ofpre- and
postautogamous injected lines were prepared as described
et al. (1987). DNA was purified from 300 pl of
by GODISKA
the NDS mixtures by adding 200 p1 of water, extracting
729
with 500 pl of phenol, reextracting the phenol phase with
500 pl of TE (10 mM Tris-HCI/l mM EDTA, pH 8.0),
treating withSevag and precipitating with 2 volumes of
ethanol for 10
in an ice bath. After washing the precip
1 1 9min
25
itate with 75% ethanol anddesiccating, the DNA was resuspended in TE.
ParameciumDNAblots:
DNA (5 pg) was
Cut
with
HindIII, separated on a 0.8% agarose gel and transferred
onto nitrocellulose as described by MANIATIS, FRITSCH
and
SAMBROOK
(1982).
For dot-blots, postautogamy DNA (300 ng) was denatured with 0.1 volume of 3 M NaOH at 65" for 30 min,
neutralized with 7.5 M NH40Ac andtransferred ontonitrocellulose using a Schleicher and Schuell Minifold I system.
Nick translation: DNA was nick-translated to produce
probes with specific activities of 5 X 10' to 1 X 10' cpm/pg
by procedures described in MANIATIS, FRITSCHand SAMBROOK (1982).
DNA blot hybridization: Prehybridization at 42" was in
50% formamide, 5 X SSC, 1 X Denhardt's solution, 50 mM
Hepes (pH 7.0), 1 mM EDTA, and 100 pg/ml salmon sperm
DNA. Hybridization was in the same buffer containing 10%
dextran sulfate 5000. Filters were washed twice with 2 X
SSC/O.l% SDS for 10 min at 23", and eitheronce or twice
with 0.1 X SSC/O. 1% SDS for 20-30 min at 68" prior to
autoradiography.
ParameciumRNApreparation: WholecellRNA was
extracted by lysing cells in guanidine hydrochloride as previously described (PREER,PREERand RUDMAN 1981).
Paramecium RNA blots: Whole cell RNA (25 pg) and
RNA markers (BRL) were separated ona1%
agaroseformaldehyde gel. After soaking the gel in 20 X SSC and
staining with ethidium bromide to confirm equal sample
loading, RNA was transferred onto nitrocellulose (MANIATIS, FRITSCHand SAMBROOK
1982).
In vitro synthesis of RNA hybridization probes: ["PI
UTP-labeled RNA complementary to each strand of plasmid pSA2.5 was transcribed in vitro using conditions similar
to those described by MELTONet al. (1984). One microgram
of linearized DNA templates was transcribed in a volume of
20 p1 containing 40 mM Tris, pH 8.0, 8 mMMgC12, 2 mM
spermidine, 25 mM NaCI, 10 mM dithiothreitol, 500 p~ of
ATP, CTP and GTP, 10 p~ UTP, 7.5 p~ [52P]UTP(800
Ci/mmol), and 13 units of T7 RNA polymerase or 10 units
of T 3 RNA polymerase. Synthesis was at 37" for 60 min.
The mixtures were then treated with 15 units of RNAsefree DNAse (Pharmacia) for 10 min at37", phenol-extracted,andthe
RNA ethanol precipitated with carrier
tRNA. Each probe had a specific activity of about 5 X 10'
cpm/pg.
RNA blot hybridization: Prehybridization wasin 50%
formamide, 5 X SSPE, 0.1% SDS, 5 X Denhardt's solution,
1 mM EDTA, 200 pg/ml salmon sperm DNA and 100 pg/
ml tRNA at 55". Hybridization wasin the same buffer
containing 10%dextran sulphate 5000and 2.5 X Denhardt's solution. The nitrocellulose was washed twice with
2 X SSC/O. 1% SDS, once with 1 X SSC/O.l% SDS, and
once with 0.1 X SSC/O.l% SDS, each at 65" for 20 min.
Primer extension: Primer extension analysis was carried
out essentiallyas described by SAMBROOK,
FRITSCHand
MANIATIS(1989). Whole cell RNA (10 pg) was hybridized
with a 20 nucleotide primer complementary to A gene
mRNA sequences from positions +62 to +43. The primer
extension reaction was then electrophoresed in a 6% polyacrylamide/8 M urea gel and autoradiographed.The labeled
products were compared to chain termination sequencing
reactions using the above mentioned primer to precisely
determine the extension product size.
H.JessopMurray et al.
730
Pre-autogamy
serotype
TABLE 1
Microinjection of plasmid pSA12.8
Preautogamy
DNA Injected
Postautogamy
Total lines
Total lines
tested Serotype tested Serotype
pSA12.8
(-264
to 11825)
68
12 A+
12
56 A- 6 A2+4
Uninjected
48A-
48
12
Q)
n
40
1 1 A+
z :
c
+
4 4
1
I
a a a 8 - 0
2
3
4
F
a
o z' Dso 0y m
5
6
7
P
8
.
18 A-
12A-
RESULTS
Injection ofpSA12.8: Previous workers have shown
that microinjection of plasmid pSA14SB
into the macronucleus of d48 causes the cells to express serotype
A both before and afterthe next autogamy (KOIZUMI
and KOBAYASHI 1989). To extend these results we
microinjected an upstream deletion derivative of
pSA14SB, plasmid pSA12.8 (seeFigure l), toobserve
whether it could also induce the mutant cells to express serotype A. pSA12.8 has 264 bp upstream of
the presumed ATGstart of the A protein while
pSA 14SB has1590 bp. Previous work in our laboratory has shown that pSA12.8 contains the minimal
upstream sequences necessary for a fully functional
promoter, as well asthe entirecoding region of the A
gene (L.D. MARTIN,unpublished data).
Throughout this report, we use two terms to describe cells that have been successfully microinjected
with plasmid DNA. Prior to autogamy, injected animals expressing serotype A and/or shown to contain
autonomously replicating cloned A genes or fragments
of A genesin their macronucleus are said to be
TRANSFORMED. After autogamy, the cloned DNA
is lost and cells subsequently able to express serotype
A due to thepresence of macronuclear copies of the
A gene, are said to be RESCUED.
The results of microinjecting supercoiled pSA12.8
into the macronucleus of d48 are shown in Table 1.
Preautogamy transformation was scored initially by
testing for serotype A expression. Of 68 injected cells,
12 gave riseto transformed lines. Twelve A+ linesand
24 of 56 A- lines were serotype tested every day
during subculturing for a further 12- 16 fissions (see
MATERIALS AND METHODS).
It was found that 7 of the
12 A+ lineslost the ability to express A prior to
autogamy. However, once through autogamy 11 of
the 12 transformed lines were rescued, and surprisingly, so were 6 of the 24 preautogamy A- lines.
Preautogamy stocks at 14" of these unusual lines were
expanded and whole cell DNA was extracted to determine the presence of the injected plasmid. Uninjected d48 acted as a control in the microinjection
experiment; no spontaneous reversion to the wild type
phenotype was observed on subculturing the cells
through autogamy (Table 1).
= 3::
-
2.3
2.2
1.4
I0 . 8
-
0.7
0.5
FIGURE
2.-Hybridization of labeled pSA12.8 DNA to HindIII
digested whole cell DNA (5 pg) isolated from preautogamy d48
animalsinjected with pSAl2.8. All lines were rescued postautogamy
to serotype A. Lanes 1 and 2: lines that showed stable serotype A
expression preautogamy. Lane 3: a line initially expressing serotype
A that lost expression immediately prior to autogamy. Lanes 4 and
5: lines that did not express serotype A preautogamy. Lane 6: wildtype DNA. Lane 7: uninjected d48. Lane 8: pSA 12.8 digested with
HindIII. Lanes 1 to 6 were shown to have equal loading of DNA
by ethidium bromide staining of the gel. Lane 7 contained less
material. Numbers to the right of the blot refer to the sizes in kb
of the HindIII fragments of pSA12.8 DNA which sewed as size
markers.
In all cases, linesrescued postautogamy were shown
to contain plasmid pSA12.8 DNA preautogamy. As
described above, some of the transformed lines did
not express A surface antigen as determined by the
serotype assay. DNA (5 pg) from several such preautogamous A+ and A- lines was cleaved with Hind111
and probed with labeled pSA12.8 DNA after Southern blotting (Figure 2). The restriction pattern obtained from transformed cellDNA(lanes1-5)
was
identical to thatof the cleaved plasmid pSA12.8 DNA
(lane 8), rather than to that
of wild-typemacronuclear
A genes (lane 6). The results show that in lines unable
to express serotype A pre-autogamy (lanes 4 and 5),
there are fewer copies of plasmid DNApresent than
in transformed A expressing cells (lanes1 and 2). This
low copy number is presumably insufficient to allow
for serotype expression pre-autogamy, but sufficient
for rescue to occur postautogamy. Lane 3 shows DNA
from a line that initially was A+, but lost expression
prior to autogamy. The DNA sample was obtained
from animals that were 20-22 fissions postinjection
when A expression was lost. However, plasmid DNA
was present in this line at a copy number similar to
that in lines unable to express serotype A preautogamy
(lanes 4 and 5). It is possible that more plasmid DNA
Rescue of a non-Mendelian Mutation
was present initially, allowing for A expression, and
that a decrease inplasmidcopy
number occurred
during subsequent subculturing. One important conclusion that can be made from these unusual lines is
that theinjected plasmid, but not high-level A expression prior to autogamy, is required for rescue.
Analysis ofthe DNA from postautogamous rescued
cell lines showed that wild-type macronuclear chromosomes containing the A serotype gene were present, rather than cloned A genes (results not shown).
DNA requirements for d48 rescue: We cloned a
variety of restriction fragments of the A gene into the
plasmid vector pT7/T3-18 to observe whether specific fragments were
capable
of rescue.
Unlike
pSA12.8, these subclones lacked transcriptional and
translational information required for A gene expression and thereforecould not transform d48 to express
serotype A. The structure of the subclones is shown
in Figure 1.
Plasmid DNA was cleaved with restriction enzymes
to free the A gene sequence containing inserts from
the vectors before injection. This was done to ensure
that theorientation of the fragments within the vector
would not interfere with their ability to rescue d48.
Because preautogamy transformation could not be
scored by A expression, the DNA ofeach injected line
was analysed by dot-blot hybridization. The blots were
probed with nick-translated fragments of the A gene
to assess the presence of plasmid, and corrected for
loading errors using a probe for the Paramecium atubulin gene. Rescue post-autogamywas measured by
the ability ofthe cells to express serotype A, asbefore.
In one experiment, plasmids pSA5.5 and pSA2.8
(see Figure 1) were compared for their ability to
rescue d48. pSA5.5 contains a fragment of the A gene
from +13 to +5482 of the coding region. The cloned
fragment inpSA2.8 carries +403 to +3175 of the
coding region. The results of the injections are shown
in Table 2. Both plasmids were efficiently established
in 20 out of about 100 injected cells. The copy number of each plasmid varied within
the 20 lines, but the
range of copynumbers was similar (results not shown).
Any differences between post-autogamous rescue efficiency was therefore not due to one plasmid being
more efficientlyestablished in the preautogamous
cells than the other. The postautogamy serotype parameter “% A+” represents the result of the serotype
assay, a score of 100 meaning that all the test animals
were immobilizedwith antiserum, 40 meaning that
only 40% of the test population were immobilized,
and so on. Any line exhibiting a value between 1 and
40% A+ was designated a “mixed culture”(HARUMOT0 1986; RUDMAN
et d . 1991). We observed that
if animals were isolated
from these postautogamy populations and subcultured, some of the secondary lines
were capable of expressing A and others were not
73 1
(data not shown). It appears that in these mixed cultures only a fraction of the progeny of one injected
cell was rescued and acquired the ability to produce
normal macronuclei. We do not know whythis occurs,
but presumably rescue in any individual animal will
depend upon the plasmid copy number in that cell at
autogamy and/or the efficiency of rescue of
the A
gene fragment itself, mixed cultures resulting when
either one is low.
The data in Table 2 show that 19 out of the 20
lines transformed with plasmid pSA5.5were rescued,
with a majorityof the postautogamy lines showing
between 40 to 100% A+. Of the remaining 80 lines,
three showed a low percentage of A expression and
this probably represents spontaneous reversion of the
mutant d48 cells to wild type, or alternatively transformation with undetectable levels ofplasmid. In comparison, only one of the 20 lines transformed with
plasmidpSA2.8 was rescued to A expression, and
none of the remaining lines were able to express A.
Uninjected controls showed no reversion to wild-type
cells. The conclusion that can be drawn from the
injection of pSA5.5and pSA2.8 is that specific A gene
sequences found in the 5.5-kb fragment are responsible for efficient d48 rescue and that neither A expression nor intact mRNA preautogamy is required for
rescue.
T o characterize the sequence requirement further,
three other subclones wereinjected. The fragment in
plasmid pSA4.5 has 1 kb downstream removed compared to the 5.5-kb fragment, and contains +13 to
+45 17 of the coding region (see Figure 1). Plasmids
pSA3.O and pSA2.5 are subclones ofthe 5.5-kb fragment cleaved at +2971. The upstream portion,
pSA3.0, consists of +13 to +2971 of the coding region, and the downstream portion, pSA2.5, contains
+2971 to +5482. The results of the injections are
shown in Table 2. All three plasmids were established
in injected cells with similar efficiency preautogamy
(data not shown). Post-autogamy, pSA4.5was able to
rescue d48 efficiently, with most ofthe rescued lines
showing a high percentage of A expression (between
40 and 100% A+). pSA3.O rescued the mutant with
poor efficiency, a majority of the lines giving a weak
postautogamy serotype result of 20% A+ and lower.
On the other hand, pSA2.5 gave an “intermediate”
type of rescue reaction with half ofthe postautogamy
lines expressing between 1 and20% A+, and the other
half expressing between21 and 60% A+. Withall
three plasmids post-autogamy rescue correlated with
the presence of injected DNA pre-autogamy. These
results indicate that specific sequences of the A gene
have the ability to rescue d48 to different extents,
some being more efficient than others.
When the results of microinjectingplasmids pSA5.5
and pSA4.5 are compared (Table 2), it can be seen
732
H. Jessop-Murray et al.
TABLE 2
Frequency of transformation and rescue from microinjection of cloned A gene fragments
Postautogamy
Preautogamy
Serotype (% A+)
Experiment
No.
Total lines
DNA
tested
lniected
Plasmid
1-20
21-40
41-60
61-80
81-100
%Rescue
~
la
Ib
IC
2a
2b
pSA2.52c
2d
pSA5.5
19/20 (7I 3 to 5482)
3
3
pSA2.8 (403 to 3 175)
Uninjected
pSA4.5 (1 3 to45 17)
pSA3.O
(1
3 to 297 1)
(2971 to 5482)
Uninjected
3100
I02
32
54
63
56
49
3
20+
80-
3
0
0
0
0
= 95
3/80 = 4
20+
82-
1
0
0
0
0
0
0
0
0
0
1/20 = 5
0/82 = 0
32-
0
0
0
0
0
0/32 = 0
23+
31-
1
1
6
0
0
23/23
6
0
= 100
0/31 = 0
0
9
0
17+
46-
16
1
1
0
0
0
0
0
0
0
17/17 = 100
1/46 = 2
24+
32-
135
0
6
1
0
0
0
0
0
24/24 = 100
1/32 = 3
49-
0
0
0
0
0
0/49 = 0
that the two are essentially indistinguishable in their
ability to efficiently rescue d48. Plasmid pSA2.5 gives
an intermediate extent of rescue, pSA3.0 a low but
detectable rescue, and pSA2.8 is unable to rescue the
mutant.
One concern was that the serotype assay did not
reflect the molecular status of the rescued lines, and
that even though the cultures were expressing low
percentages of A, the cells actually had a wild-type
copy number of A genes in their macronuclei. To
address this concern, purified DNA from a random
selection of post-autogamous animals from each serotype group and from each injection experiment was
analysed by dot-blot hybridization. Duplicate blots
were probedwith the A gene andwith a probespecific
forthe Paramecium a-tubulingene. The A genespecific hybridization signal was normalized to that of
a-tubulin to calculate the A gene copy number. The
relationship between the extent of the serotype reaction and the A gene copy number is shown in Figure
3. A positive correlation is observed between A gene
copy numberandtheserotypereaction.
T h e data
underscore the fact that pSA5.5 and pSA4.5 can
rescue d48 with high efficiency, while pSA2.5 rescues
with “intermediate” efficiency and pSA3.O with poor
efficiency.
It is unknown why lines rescued by injection of
plasmids pSA4.5, pSA2.5 and pSA5.5 expressed such
a wide range of postautogamy serotype levels. We
examined the possibility that this range in serotype
levels was due to variation in the preautogamy copy
number of the injected plasmid among the different
lines. N o such correlation was observed when dotblots containing pre- and post-autogamy DNA from
FIGURE3.-Relationship between copy number of A genes and
extent of serotype expression in randomly selected postautogamy
lines. The lines were generated from individual cells injected with
the five plasmids shown
to the right of the graph.The copy number
of A genes present in the various lines was determined by DNA blot
hybridization.
lines injected with plasmids pSA4.5, pSA3.O and
pSA2.5 were analysed (results not shown). Thus, the
pre-autogamy plasmid copy number in a population is
not linearly related to the extent of rescue observed
in the postautogamy population. Whethera linear
relationship exists at the cellular level between copy
number of injected plasmid and post-autogamy serotype response requires analysis of individual cells carried through autogamy.
RNA analysis: KOIZUMI and KOBAYASHI (1989)
concluded that a factor responsible for inclusion of
the A gene into the newly forming macronucleus is
present in the cytoplasm of autogamous and conjugating wild-typecells.We
examined the possibility
that a stable RNA from the A gene region might play
a role in this process.
Whole cell RNA was extracted from wild-type cells
Rescue of a non-Mendelian Mutation
1 fiss
2 fiss
past
autog
log
autog
autog
"
"
co
co
co
co
w
d
d
d
~ ~ u a o u ~ n u a n u
1 2 3 4 5 6 7 8 9101112
= 9.5
"7.5
-
4.4
= 2.4
= 1.5
-
0.2
733
prepared from d48 (Figure 4, lanes 2, 3, 5, 6, 8, 9,
11, 12).At longer exposures both A gene mRNA
species were faintly detectable in cells stably expressing serotype D (data not shown). Primer extension
analysis using RNA isolated from A-gene expressing
cells indicated a single 5'end of the Amessage located
seven bases upstream from the ATG presumed to be
the initiation codon (results not shown). It is possible
thatthe IO-kbspecies represents a different conformer of the 8.5-kb species, or alternatively is a result
of heterogeneity in transcriptional termination. No
transcripts smaller than full-length mRNA that could
be candidates for the factor were detected with this
highly sensitiveprobe in any ofthe RNA preparations
(Figure 4), even when longer autoradiographic exposures were examined. When the RNA probe that is
not complementary to theA gene message was hybridized to the blot, no signal was observed in any of the
whole cell RNA extracts (results not shown), demonstrating thatno stable "antisense" RNAis present. We
were therefore unable to detect a stable RNA cytoplasmic factor.
DISCUSSION
FIGURE4.-Hybridi~ation of an RNA probe complementary to
a portion of the A gene mRNA with Paramecium whole cell RNA.
The labeled probe was synthesized by in vitro transcription of
plasmid pSA2.5 as described in MATERIALS AND METHODS. Whole
cell RNA was extracted from wild-type animals expressing serotype
A (lanes 1 , 4, 7, lo), serotype D (lanes 2, 5, 8, 11). and from d48
cells (lanes 3 , 6 , 9 , 12). RNA preparations were made from animals
in various growth and developmental stages including log phase
(lanes 1 , 2, 3), autogamy (lanes 4, 5, 6), one fission past autogamy
(lanes 7, 8 , 9). and two fissions past autogamy (lanes 10, 1 1 , 12).
The Northern blot was exposed to X-ray film for 10 min at room
temperature. Numbers to the right of the blot refer to the sizes in
kb ofmarker RNAs (BRL).
expressing serotype A and D, and from d48 cells.
RNA preparations were made from cells in log phase,
autogamy, one fission past autogamy and two fissions
past autogamy. The RNA was electrophoresed, blotted onto nitrocellulose and separately hybridized with
twolabeledRNA
probes complementary tothe
strands of plasmid pSA2.5. The RNA probes were
generated by T7/T3 RNA polymerase transcription
in vitro as described in MATERIALS AND METHODS.
The probe complementary to the A gene mRNA
hybridized to twoRNAspeciesisolated
from wildtype cells expressing the A gene (Figure 4, lanes 1, 4,
7, 10). These two RNA species were present in a p
proximately the same relative abundance in RNA
preparations from wild-type cells at different growth
and developmental periods.One species was 8.5 kb in
length, which is identical to thesize ofA gene mRNA
reported by PREER,PREERand RUDMAN(1981). A
second hybridizing species was roughly 10 kb. The
probe did not hybridize with RNAfrom cells expressing serotype D after shortexposure times, or to RNA
The non-Mendelian pattern of inheritance shown
by d48 represents a macronuclear deficiency that
prevents proper processing of micronuclear A genes
into the newly forming macronucleus at autogamy.
Transformation and subsequent rescue by microinjectionof a plasmid containing the A gene intothe
macronucleus of d48 shows that thed48 phenotype is
related to the absence of the A gene in the old macronucleus (KOIZUMI
and KOBAYASHI1989). KOIZUMI
and KOBAYASHI(1989) also showedthat acytoplasmic
factor produced during autogamy was responsible for
proper inclusion ofthe A gene into the newly forming
macronucleus. Taken together, these results suggest
that in wild-type cells, the A gene in the old macronucleus ensures the presence of a cytoplasmic factor
that is responsible for the properprocessing of the A
gene at autogamy. In d48 cells, where there are few,
if any intact copies of the A gene in the old macronucleus (EPSTEINand FORNEY
1984), deletions occur in
the newly forming macronucleus at autogamy.
We have defined a region of the A gene that is
sufficient to rescue d48. A 4.5-kb internal fragment
of the A gene, from +13 to +45 17 of the coding
region rescues d48 as efficiently asa plasmid carrying
the intact gene. Thus, rescue of d48 does not require
upstream transcriptional control sequences, intact A
mRNA or A serotype protein. An unexpected result
was the "intermediate" extent of rescue obtained with
the 2.5-kb downstream A gene fragment (from +297 1
to +5482 of the coding region). Combining the results
of microinjection of the 4.5- and 2.5-kb fragments
indicates that the region +297 1 to +4517 of the A
734
H. Jessop-Murray et al.
gene contains critical sequences. However,the 3.0-kb
“upstream” fragment (from +13to+2971
of the
coding region) gave a low but detectable levelof
rescue. Comparing this fragment to the2.8-kb portion
(from +403 to +3175) whichwas not capable of
rescuing d48, suggests that the region +13 to +403
may also contribute to the rescue process.
Recently, YOU et al. (1991) published a report relevant to work reported here. They showed that microinjection of an 8.8-kb fragment internal to the A
gene rescued d48. The 4.5-kb fragment reported here
is internal to the 8.8-kb fragment.
The molecular mechanism for the involvement of
a region of the A gene in the old macronucleus with
DNA processing of the micronuclear A genes is unknown. We found no evidence for asmall, stableRNA
that could either represent or encode a cytoplasmic
factor. However, we cannot rule out the possibility
that a short-lived, unstable RNA may be synthesized
at autogamy.
Alternatively, the injected DNAitself might act
directly as the factor. At autogamy, when the macronucleus is degenerating, fragments of the A genes may
be liberated into the cytoplasm. These could act in a
variety ofways. For instance, they may sequester a
processing factor that if left unbound cleaves A genes
in the newly forming macronucleus upstream of the
coding region. Some liberated A gene fragments may
have a greater affinity for the processing factor than
others, thereby sequestering it more efficiently. This
could explain why the cloned A gene fragments injected into d48 either showed full, intermediate, or
very poor rescue. Alternatively, the released A genes
may act directlyon DNA processing ina yet unknown
manner.
The unusual type of genetic behavior manifested
by d48 is not restricted to inheritance of the A gene.
Other traits in ciliates show similar patterns of inheritance, such as mating type and trichocyst mutants in
P. tetruureliu (SONNEBORN
1975; SONNEBORN
and
SCHNELLER
1979), and surface proteins in Tetruhymena thermofihilia (DOERDER
and BERKOWITZ1987).
In these three cases, the old macronucleus is involved
in the passageof information about itsmolecular
status to the newly developing macronucleus at autogamy and conjugation. The presence or absence of
specific DNA sequences inthe old macronucleus may
be responsible for controlling these traits.
We thank TIMFITZWATER
for his technical assistance with the
in vitro synthesis of RNA probes. The research reported here was
supported by National Institutes of Health grant GM 31745-08.
LITERATURECITED
DOERDm,F.P., and M. S. BERKOWITZ,
1987 Nucleocytoplasmic
interaction during macronuclear differentiation in ciliate protists: genetic basis for cytoplasmic control of SerH expression
during macronuclear development in Tetrahymena thermophilia.
Genetics 117: 13-23.
EPSTEIN,L.N., and J. D. FORNEY,1984 Mendelian and nonmendelian mutations affecting surface antigen expression in
Paramecium tetraurelia. Mol. Cell. Biol. 4 1583-1590.
FORNEY,
J. D., and E. H. BLACKBURN,
1988 Developmentally
controlled telomere addition in wild-type and mutant Paramecia. Mol. Cell. Biol. 8: 251-258.
GILLEY,D., J. R. PREERJR., K. J. AUFDERHEIDE
and B. POLISKY,
1988 Autonomous replication and addition of telomerelike
sequences to DNA microinjected into Paramecium tetraurelia
macronuclei. Mol. Cell. Biol. 8: 4765-4772.
GODISKA,R., K. J. AUFDERHEIDE,D.GILLEY,P. HENDRIE,T .
FITZWATER,
L. B. PREER,B.POLISKY and J. R. PREER,JR.,
1987 Transformation of Paramecium by microinjection of a
cloned serotype gene. Proc. Natl. Acad. Sci. USA 8 4 75907594.
HARUMOTO,
T., 1986 Induced change in a non-Mendelian determinant by transplantation of macronucleoplasm in Paramecium
tetraurelia. Mol. Cell. Biol. 6 3498-3501.
KOIZUMI,
S., and S. KOBAYASHI,
1989 Microinjection of plasmid
DNA encoding the A surface antigen of Paramecium tetraurelia
restores the ability to regenerate a wild-type macronucleus.
Mol. Cell. Biol. 9: 4398-4401.
MANIATIS, T., E. F.FRITSCH and J. SAMBROOK,
1982 Molecular
Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
MELTON,D. A., P. A. KRIEG,M. R. REBAGLIATI,
T. MANIATIS,
K.
ZINNand M. R. GREEN,1984 Effxcient in vitro synthesis of
biologically active RNA and RNA hybridization probes from
plasmids containing a bacteriophage SP6 promoter. Nucleic
Acids Res. 12: 7035-7056.
PREER,
J. R., JR., 1986 Surface antigens in Paramecium, pp. 301339 in Molecular Biology of the Ciliated Protozoa, edited by J. G.
GALL.Academic Press, New York.
PREER,J. R., JR., L. B. PREERand B.M. RUDMAN,
1981 mRNAs
for the immobilization antigens of Paramecium. Proc. Natl.
Acad. Sci. USA 78: 6776-6778.
RUDMAN,
B., L. B. PREER,B. POLISKYand J. R. PREER,JR.,
1991 Mutants affecting processing of DNA in macronuclear
development in Paramecium. Genetics 129 47-56.
SAMBROOK,
J., E.F. FRISCHand T. MANIATIS,1989 Molecular
Cloning, A Laboratosy Manual, Ed. 2. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
T. M., 1975 Paramecium tetraurelia, pp. 469-594 in
SONNEBORN,
Handbook of Genetics, Vol. 2, edited by R. KING. Plenum, New
York, N.Y.
SONNEBORN,
T. M., and M. V. SCHNELLER,
1979 A genetic system
for alternative stable characteristics in genomically identical
homozygous clones. Dev. Genet. 1: 21-46.
You, Y., K. AUFDERHEIDE,
J. MORAND,
K. RODKEY
and J. FORNEY,
1991 Macronuclear transformation withspecificDNA fragments controls the content of the new macronuclear genome
in Paramecium tetraurelia. Mol. Cell. Biol. 11: 1133-1 137.
Communicating editor: S. L. ALLEN