Copyright 0 1991 by the Genetics Society of America

Bertina Rudman, LouiseB. Preer, Barry Polisky and John R. Preer, Jr. Program in Molecular, Cellular and Developmental Biology, Department of Biology, Indiana University, Bloomington, Indiana 47405

Manuscript received March 18, 1991 Accepted for publication May 17, 1991 ABSTRACT In Paramecium tetraurelia, stock 51, the A surface protein is coded by the wild type AI’ gene, present in micronuclei in two copies and in macronuclei in about 1500 copies. DNA processing, comprised of DNA cleavage, copy number amplification and telomere addition occurs at autogamy and conjugation when old macronuclei degrade and new macronuclei are formed from micronuclei. In this paper we characterize mutants with macronuclear A gene deletions. These mutants are notable in three respects. First, the mutants do not appear to be simple micronuclear deletions. Although genetic analysis shows that the d l 2 mutant d l 2(-1300) is homozygous for the allele A-”” and the mutant d 12(+ 1) for A+’, analysis by the polymerase chain reaction indicates that the micronucIei in these two mutants contain intact, but presumably altered, micronuclear A genes. They undergo deletion during DNA processing when new macronuclei are formed. Second, the position of the deletions in these alleles has been shown to change. The deficiency present in the d l 2 allele A”3w was originally determined to extendfrom position - 1300 (relative to the startof translation of the A gene) to theend of the chromosome. Later, a derivative of this strain, homozygous for the d l 2 allele A+’ was isolated in which the start site of the deletion was found to have moved from - 1300 to 1. Third, a surprising interaction occurs in crosses between a line homozygous for the d l 2 allele and one homozygous for the wild-type A’’ allele. Previous work on thenon-Mendelian d48 mutant (which has intact A’’ genes in its micronucleus, but has truncated A’’ genes in its macronucleus) has shown that intact A” alleles must be present in the old macronucleus in order for As’ alleles to undergo proper processing. We find that d l 2 alleles act on A5’ allelesin heterozygotes such that intact macronuclear A genes are no longer required for proper processing of A”. Thus, in crosses of 51 X d l 2 (either +1 or -1300) d l 2 exconjugants, as well as 51 exconjugants, give rise to clones carrying both intact A’’ and truncated d l 2 alleles. Remarkably the d l 2 alleles, which are themselves deleted during processing, are capable in the heterozygote of fostering normal processing of the As’ allele.

+

T

HE A gene that determines surface protein A in wild-type strain 51 of Paramecium tetraurelia is found near the end of a macronuclear chromosome oriented with the 3‘ end of the gene nearest a telomere (FORNEYet al. 1983). Two copies of the gene are present in the diploid micronucleus and about 1500 copiesin the macronucleus. In the non-Mendelian mutant,d48,the intact A gene is present in the micronucleus, but was reported tobe absent from the macronucleus (EPSTEINand FORNEY1984). EPSTEIN and FORNEY(1984) showed that the macronuclear deletion in d48 starts in the vicinity of 1, measured from the start of translation. Subsequently, in four separate cloned genes, DNA sequencing revealed that the deletions started at -79, +33, +121 and +149 (FORNEYand BLACKBURN 1988) with a telomere attached at the end of each cloned gene. Since the A gene is missing,serotype A cannot be expressed under conditions known to produce A andthe strain is designated A-. EPSTEINand FORNEY(1984) reported that when d48 (A-) is crossed to 51 wild type (A+)the d48 exconjugant usually produces an A- F1 clone and

+

Genetics 1 4 9 47-56 (September. 1991)

the 5 wild-type 1 exconjugant produces an A+ F1clone. No further change in type occurs after autogamy in the F2 generation. Although halfof the F2 clones derived from the 5 1 wild-type exconjugant are homozygous for theA allele that came from the d48 parent, all of the F2 clones of the 51 exconjugant are wildtype A+. These results demonstrate that the micronuclei of d48 must contain the complete wild-type A gene, A5’. However, the non-Mendelianpattern of inheritance shown by d48 does not indicate true cytoplasmic inheritance but appears instead to be due to the influence of the old macronucleus on the newly forming macronucleus during conjugation and autogamy. This conclusion is reached on the basis of a series of transfers of macronucleoplasm and cytoplasm by HARUMOTO (1986) and KOIZUMI and KOBAYASHI (1989). Additional workhas indicated that the macronuclear element responsible for this effectis actually the A gene itself. In 1985, TERUE HARUMOTOin our laboratory (personal communication) obtained data suggesting that injection of a portion of the cloned A

48

B. Rudman et al.

gene into the macronucleus of d48 would cause permanent reversion of d48 to wild type. KOIZUMI and KOBAYASHI989) (1 showed that injection of the complete A gene into the macronucleus of d48 results at the next autogamy in the permanent restoration of the ability of the cells to produce A. More recently, YOU et al. (1991)and H. JESSOP-MURRAYand L. MARTIN (personal communication) have shown that injection of specific subfragments of the A gene into d48 results in permanent rescue. Thus, old macronuclei bearing at least a portion of the A gene must be present forproper DNA processing of new A genes during development of new macronuclei. Since d48 is deficient in complete macronuclearA genes, proper processing of new A genes at conjugation and autogamy does not occur. Possibly, d48 arose as a consequence of a developmental abnormalityin DNA procA genes essing. Once lost fromthemacronucleus, could not be restored at subsequent autogamies because the A gene, necessary for correct processing, would be missing. This dependence of new macronuclear A genes on the presence of old macronuclear A genes constitutes an anomalous genetic mechanism which appears to account for the facts acquired thus far. Its molecular mechanism is completely unknown. Inaddition to the non-Mendelian mutant,d48, which requires the presence of A genes in the old macronucleus for proper A gene processing, mutants d 1 and d 12were isolated by L. EPSTEINand J. FORNEY (unpublished results). These mutants also lack complete A genes in their macronuclei, and preliminary results suggested that these mutantswere inherited in a Mendelian fashion. In this paper we have characterized the d l and d l 2 mutants, and show that there are actually two d l 2 strains that have mutant alleles at the A locus. T h e d l 2 strains exhibit both Mendelian and non-Mendelian inheritance. We show that in heterozygotes carrying eitherof the d l 2 alleles and thewild-type allele, processing of the wild type allele is usually normal. Normal processing of the wild-type allele occurs not only in cells whosemacronuclei have normal A genes, but also in cells whose macronuclei have defective A genes. Finally, we present evidence that the d l 2 mutant micronuclear A gene copies are not truncated, but that they affect DNA processing by causing deletions during the formation of new macronuclei during autogamy and conjugation. MATERIALS AND METHODS

Strains: Stock 51 (ATCC #30303) is homozygous for the wild-type A'' gene. Strain d48 was derived from 51as (1984) using Xdescribed earlier by EPSTEINand FORNEY ray mutagenesis and antiserum selection. Strains d12, d l and d l 6 were obtained in a similar fashion. Several amicronucleate lines, and also chimeric strains produced by transplanting wild type micronuclei from stock 51 into amicronucleate d12, were provided by S. KOIZUMIand S. KO-

BAYASHI. They removed micronuclei from a number of d l 2 cells, designating them K1, K2, etc. They then transplanted wild-type micronuclei into these cells, and used each to start new lines, Kl-I, K1-2, K2-1, etc. d l , d l 2 or d48 cannot produce serotype A surface antigen when cultured at 34" for several fissions. By contrast, wild-type stock5 1,cultured at34", generally switches to serotype A within 3 or 4 fissions. The original d l 2 was later redesignated d 12(- 1 300) to denote its deletion start relative to thebeginning of translation of the A gene. A variant of d l 2(-1300) with analtered deletion appeared spontaneously in our culture collection and was designated d l 2(+ 1). These strains are characterized in more detail in RESULTS. d48 has been known to revert to wild typesporadically withlow frequency, and d l 2 has been suspected of reverting two or three times over the course of several years. The strain d46 (ATCC #30987) is isogenic with stock5 1 but homozygous for several marker genes including the A' allele (SONNEBORN 1975). Culture of paramecia: Paramecia were cultured in an infusion of 1.5 g/liter of wheat Cerophyl (Pine Brothers, Kansas City, Missouri) supplemented with 0.1 g of BactoYeast Extract, 1 mg of stigmasterol, and 0.4 g Na2HPO4.It was inoculated with Klebsiellapneumoniae a day or two before use. Crosses: Matings, induction of autogamy and serotype testing were carried outas described by SONNEBORN (1975). A cross of two lines in Paramecium yields F1 exconjugant clones withidentical genotypes. An FPobtained by inducing autogamy in such FI clones yields a series of homozygous F2 clones. For each genetic locus, 1/2 the F2 clonesare homozygous forthe allele found in oneparentand1/2 are homozygous for the allele found in the other parent.In the crosses described here at least one Mendelian marker was present, and lines were eliminated from further consideration if these markers did not indicate proper exchange of nuclei and normal genetic behavior. The Mendelian markers used were twisty, paranoic A, and a trichocyst nondischarge mutant (SONNEBORN 1975). Mating type was used as a cytoplasmic marker. Moreover, the paranoic marker also usually made it possible to distinguish the two parents, for the original phenotypes were almost always still present at the time that exconjugants were separated. Tests for the ability to produce serotype A were done by allowing cellsto undergo several fissionsat 34 *, then using antiserum to test for serotype A. The most reliable method was that employed by EPSTEINand FORNEY (1984), adding fresh medium to double the volume of cultures in test tubes daily for four days at 34" In most cases wild-type cultures were found to bemore than 50% A,usually nearly 100% A and were designated A+. Those producing no A were designated A-. Only in very rare instances was a culture thatfailed to show serotype A by this test found tohave the A gene by molecular tests. In such cases a second growth period of four days at 34" always revealed serotype A antigen expression. EPSTEIN and FORNEY (1984) reported that in the progeny of the cross d48 X 51, occasional exconjugant clones were produced which yielded both A+ and A- subclones. We have confirmed their results and refer to such clones as "mixed" clones. They also occur in progeny of the cross d48 X d12. Preparation of DNA from Paramecium: Up to 500,000 cells (producing about 75 pg DNA)were resuspended in 0.1 ml of their own culture medium and squirted into 0.2 ml of NDS (1% sodium dodecyl sulfate, 0.5 M NaaEDTA, 10 mM Tris-HCI, pH 9.5) at 65". After 48 hr at 65", 100 PI HPO were added. The lysates were phenol-extracted, treated with Sevag solution (24 parts chloroform, 1 partisoamyl alcohol) and precipitated with two volumes of ethanol for 10 min in an ice bath. After centrifugation the precipitate was washed

Mutants Affecting 49 DNA Processing

once with 75% ethanol and desiccated. Occasionally a secondphenoltreatment wasnecessary in order toobtain efficient cutting with restriction enzymes. Hybridization: Southern blots and hybridizations were carried outas previously described(GODISKA et al. 1987). Plasmids: The plasmidpSAl4SBcontainstheregion -1590 to +8229 of the A gene in a pT7/T3-18 vector (GODISKA et al. 1987). The plasmid pSA3.75Hd spans the region -3300 to +456 of the A gene and is in a pUC8 vector. It was prepared by J. D. FORNEY. Polymerase chain reaction (PCR): The reaction mixes were prepared by adding: 3 PI of purified DNA solution of the desired concentration, 5 pl of 1% NP-40, 1 pl Tetrahymena DNA (0.1 pg/pl) as carrier, 10 pl of 10 X buffer (500 mM KCI, 100 mM Tris-CI, pH 8.3, 15 mM MgCln, 0.1% gelatin (wt/vol)), 16 pl of NTPs (12.5 mM each of dCTP, dGTP, dATP and dTTP), 5 pl of each primer (OD260 = 5.0), 3 pl of Taq polymerase (total of 2 units) and Hz0 to 100 pl. When using whole cells rather than isolated DNA, the following procedure was used. A population count on the culture to be used was made by spreading 20-60 pl of the culture in a long narrow streak onto a glass microscope slide and counting the number of cells using low magnification. The volume of the culture needed to give the required number of cells was computed, and thisvolume was added to the 0.5-ml reaction tube. Then 1.7 times the volume of 1% NP-40 was added and the mixture quickly spun for 10 sec at top speedin a microfuge. T h e supernatant was removed with suction to leave a volume of 8 pl. Rapid processing is necessary to avoidpremature lysis of the cells. After 10 min at 65" the 8-p1 aliquot was used instead of 3 pl of DNA and 5 pl of 1% NP-40 in the reaction mix described above.The amplifications were made in a Perkin Elmer PCR cycler as follows. A preliminary incubation at 94" for 15 sec then 60 cycles of 92" for 1 min, 50" for 2 min, 72" for 2 min with a 3-sec extension on the72" segment, followed by a final 7 min at 72". RESULTS

A gene deletions begin at different sites in different mutants: Before investigating the genetics of the mutants other than d48 we needed to characterize the position of their deletions. L. N. EPSTEINand J. D. FORNEY (unpublished results) isolated the A- Mendelian mutant, d12, which they found, using hybridization to Southern blots, was deleted startingat approximately position -1300. In our laboratory a variant of this strain arose which proved to be inherited in the same way as the original d l 2,but which we found to be deleted starting farther downstreamat approximately position 1. T h e point of deletion appears to be very close or identical to that in d48. These two d l 2 strains are designated d12(-1300) and d12(+1). See Figure 1. The two strains can be distinguished from each otherandfrom wild-type strain51 by hybridization to Southernblots of restriction enzymedigested genomic DNA. See Figure 2. It is also possible to distinguish them by PCR using primers spanning the region from - 1361 to -5 12 (Figure 1) which give an 850-bp productwith 100 cells of d12(+1) and no product with d l 2(-1300). Experiments utilizing this technique will be described later and are shown in Figure 4.

+

In addition, a Mendelian A- mutant, d l , was isolated by L. N. EPSTEINand J. D. FORNEY(personal communication), which they found was deleted starting at approximately- 1000. We find, using Southern blots, that the deletion point in all the current lines of d l has shifted and is now found to start at approximately +1 (data not shown). Mendelian inheritance in the crosses of d l to 51 wild type:Crosses of d l 2 to wild type yield the results expected if d l 2 differed from wild type by a single recessive gene mutation. We find essentially the same results for both d12(-1300) and d12(+1). Most of the F1 exconjugant clones were A+, i.e., capable of expressing A,before they went intoautogamy, even those derived from thed l 2 exconjugant. Although a few of the F1 lines did not expressA, it is thought that the numberof fissions before autogamy is insufficient to allow adequate testing of the ability of all cultures togoto A at hightemperature.Ina cross of d12(-1300) to 51 wild type the genotype of the F1 was examined by hybridization to blots. The DNA from four of these FI lines (the exconjugants from two pairs, both of which were A+) was isolated and cut with HindIII, electrophoresed, blotted and probed with labeled pSA3.75Hd DNA. This plasmid contains the sequences upstream of the A gene from -3300 to +456. four showed two bands, the larger oneindicating the presence of the 51 wild-type allele, and the smaller one indicating the presence of the -1 300 deletion. Three of the F1 lines are shown in Figure 3, lanes 1-3. An F2 was obtained by establishing a series of lines from autogamous F1 isolations. The resulting F2 lines were then tested for their ability to produce A after four or more fissions at 34". The ratio of A+ to Alines was determined for each exconjugant clone that went through autogamy. The results are shown in Table Z and summarized in Figure 7. T h e FP pattern is clearly Mendelian, very close to the expected single factor ratioof 1 A+:1 A- both for theindividual ratios and for the overall ratio of 149: 154 summed for the crosses d12(+1) X 51 and d12(-1300) X 51. PCR using primers spanning the region from -1 361 to -512 (Figure 1) was used to determine the deletion points in the FZ lines. In the cross of d12(+1) X 51, 20 of the A- FP clones all showed the +1 deletion as expected. In the cross of d12(-1300) x 51, 12 randomly selected A- F2 clones all showed the -1 300 deletion. See lanes 1-6, Figure 4, which show some of the lines with the -1 300 deletion. It is important to emphasize, however, thatthe Mendelian pattern for both exconjugants is not what might have been expected on the basis of the hypothesis developed to explain the d48 mutant, e.g., the necessity for the presence of a sufficient number of A genes in the old macronucleus in order to obtain the normal high number of A genes in the newly forming

50

B. Rudman et al. Transcription Start -14

Transcription End 8202

+4-

-1361 -512

5107 5501

.....

..... .....)

FIGURE1.-The A gene and the mutants. The heavy bar indicates the 14 kb 4 coding region of the A gene; thelight bars, the flanking regions. There are no known genes between the 3' end of the A gene and the nearest telomere, which is about 8 kb to the right d 16 of the A gene on thediagram. Inserts intothe plasmids pSA3.75Hd and pSA14SB are indicated below the +1 map. H represents a Hind111 site. . d 48, d 12(+1), d 1(+1) . 4

3.75 kb

b

.....

-1000

-. d 1 (-1000) -..I

.....

-1300

d 12 (-1300)

macronuclei of A+ cells. On the basisof the latter hypothesis one would predict thatthe lackof the normal number of A genes in the macronucleus of d l 2 would have resulted in the production of all Aprogeny from the d l 2 exconjugant clones. However, in the heterozygous genotype, consisting of one d l 2 allele and one 51 wild-type A allele, the wild type A gene is processed normallyinto macronuclear A genes at conjugation, even in the absenceof complete A genes in the old macronucleus. We will return to a consideration of this effect later. Deletions in Mendelian mutants are determined by alleles at the A locus: A cross of d12(-1300) AX d l 2(+ 1) A- gave allA- F1 exconjugant clones. The DNA from six of these F1 lines was isolated and the DNA was cut with HindIII, electrophoresed, blotted and probed with pSA3.75Hd. Allsix preparations showedtwobands, indicating approximately equal amounts of the two parental macronuclear chromosomes. Three of the F1 lines are shown in lanes 4-6 of Figure 3. Fz lines wereobtained by inducing autogamy in the F, lines. Again all the lines were A-. Eighteen of the Fz lines chosenat random were screened as described above with blots and hybridization, in order to see how much ofthe Achromosome each contained (data not shown). It was found that nine of these lines were deleted at -1 300 and nine were deleted at +1, precisely the 1:l ratio expected on the basis of a single Mendelian factor difference. PCR analysis, using the primers spanning the region -136 1to -5 12 as described above, confirmed these results. See Figure 4, lanes 7-10. Thus, the position of the deletion in the

two strains is determined by a pair of simple Mendelian alleles. The question of whether the d l 2 mutations are allelic withthe A locus that determines the amino acid sequence of the A protein was addressed by further crosses. d l 2 was derived from stock 5 1which is homozygous for the allele A5'. The strain d4-6 is isogenic withstock 51 but homozygous for several marker genes including the AZ9 allele Fc 'NEBORN 1975). Serologically distinguishable iml:oi.ilization proteins determined by different alleles have been shown to differ in their primary amino acid sequence (reviewed in SONNEBORN 1975). Wehaveutilized appropriate antisera that can distinguish the two antigenic types. If the two d l 2 strains are mutant at the A locus, a cross of d l 2 to astrain bearing AZ9should give inthe Fz A+ and A- clones in a 1:1 ratio and all the A+ would be serotype 29A. If the two are not allelic, then the gene for serotype 5 1A should be present in d 12, since d 12 was derived from stock 5 1. Therefore, 5 A1 recombinants, as well as 29A should appear in the Fz. The data given in Table 2 show that in the cross of d12(+1) tod4-6 (containing the gene for 29A, rather than 5 A) 1 segregation ratios of 1 A+: 1 A- were found. When the individual ratios found for thiscrossin Table 2 are summed, a ratio of 59 A+:71 A- was obtained. Totals for the cross of d12(-1300) to d4-6 in Table 2, yield a ratio of 76 A+:100 A-. All A+ lines were 29A and none were 51A. The slight excess of A- lines was probably due to the fact that it is more difficult to induce 29A at high temperature than it is to induce 5 A, 1 and hence a few of the linesjudged to be A- were actually At. In fact, when the A gene in

Mutants Affecting DNA Processing

FIGURE2.-DNA blot analysis of the d l 2 deletions. DNAs ( 5 pg/lane) were cut withHind111 and electrophoresed on agarose. Lanes I and 4. d 12(- 1300). Lanes 2 and 5 , d 12(+ 1). Lanes 3 and 6. stock 51. The gelwas blotted and probed with pSA3.75Hd (spanning the region -3300 to +456) and shown in lanes 1, 2 and 3 . The blot was then washed and reprobed with pSA 14SB(spanning the region -1590 to +8229) and shown in lanes 4, 5 and 6. The 3.75Hd probe reveals the short remaining portion of d12(-1300) in lane 1 and the longer piece of d l 2(+1) in lane 2. The latter terminates near a Hind111 site and hence appears fuzzy because of variation in the precise cut site and variation in the length of telomeres. The pSA14SB probe reveals all the internal fragments of the A gene in stock 5 1 which are missing in the two mutant deletion strains as well as a few weaker cross-reacting bands derived from regions of the genomeother than the A gene. The differences i n the cross-hybridizing bands (approximately 8 kb) in the two d l 2 mutants are not specific for the mutants.

one of these lines judged to be A- on a routine test was examined by PCR, the results indicated that the complete A gene was present. Subsequent serotype testing revealed that it was indeed able to produce 29A. Thus,there is no evidence forthe presenceof additional independent loci that affect the deletions in any way. The only interpretationthat we find compatible with these results is that d12(-1300) is homozygous for the allele A-””” and that d 12(+ 1) is homozygous for the allele A+’. Mendelian non-Mendelian inheritancein crosses of dl2 and d48: We find (see Table 1 and summary in Figure 7) that thecrosses of d48 X d 12 (both- 1300 and +1) often show a simple Mendelian pattern of inheritance in the F2 (1:1 ratio of A+:A-). However, we also see that in many cases the ratios are strongly biased in favor of the A- class. These biased ratios are derived not only from the d48 exconjugant but also from the d l 2 exconjugant. Thus, the non-Mendelian effect arising from adeficiency of intact macronuclear A genes is characteristic not only of d48, but also of

51

FIGURE3.-DNA blotanalysisof heterozygotes. DNA (2 pg/ lane) was extracted from F, cultures, cut with Hindlll, and electrophoresed on agarose. The gel was blotted and probed with pSA3.75Hd. The DNAin lanes 1-3 was taken from different exconjugant clones of the cross d l 2(-1300) X 5 1. The DNAin lanes 4-6was taken from the cross d12(-1300) X d12(+1). The band of about 2.0 kb represents the sequences present in the d l 2(-1300) chromosome. The band at approximately 3.8 is derived from wild-type chromosomes in lanes 1-3. and from the d12(+1) chromosomes in lanes 4-6.

d 12.KOBAYASHIand KOIZUMI(1990) reported results similar to these, except that they occasionally found FI exconjugants that failed to segregate in the FP,all progeny remaining A+. They also reported such lines from the cross d l 2 x 51. We have never observed such clones. Perhaps such lines represent reversion of d l 2 to wild type. It is not knownin their crosses whether they were working with d12(+1) or d 12(- 1300), since the crosses predated the discovery of the two types of d 12. Although in the data reportedin Table 1,deviations from a 1 :1 segregation appeared more frequently in the cross d48 X d 12(- 1300) than in the cross d48 X d12(+1) we cannot be sure that d12(+1) and d 12(- 1300) really differ in this respect, for considerable variation was found in the results from crosses made at different times. In addition, it appears that the d48 exconjugant is more likely to yield aberrant results than the d l 2 exconjugant, although this conclusion is also not certain. In the cross of d l 2(-1300) X d48 one half the autogamous FP clones should have the genotype A’‘/ A’’ and the other half the genotype A”300/A-’3no, In some cases (see Table 1) the progeny of the exconjugants are all A-. In these exceptional cases, the A’’/ A” segregants are A- and their macronuclei should have deletions. Consequently we might expect them to be identical to d48. Theyarose in a non-Mendelian fashion because their macronuclei were developed in cells (the d 12 and the d48 exconjugants) with reduced numbers of A genes in their macronuclei. If PCR were

B. RudnQanet al.

52

A- F2

TABLE 1

Fn ratios from crosses involving 51,d48 and d l 2

-;io

Progeny of 5 I Progeny of d 12 exconjugant exconjugant Cross

A':A-

F2

d12(-13M))x51 d12(-1300)xd12(+1)

::n r-~

1 2 3 4 5 6 7 8910M

/\+:A-

24:24 5 1 A21:16 * X d12(+1)A28:20 18:18 Sun1 = 9 I :78 6:6 5:7 6:6 5:7 4:8 2:10 6% 7:13 Sum = 58.76

51A' X d12(-1300)A8:4

7:5

1 .o

0.5

Sum for both crosses = 149: 154 Progeny of d4A exconjugmt

d48A-

X

d 12(+ 1)A-

Progeny of d I2 exconjupnt

A+:A-

A':A-

11:11 11:13 19:13 13:l 1 15:16

7:7 10:14 12:13 2522 21:17 18:17

19:22 0:48

0:15

21:15 d48A-

X

d12(-1300) 10:14

5:19

14:10

2:20 0:23 6:17

13:10

6:18

3:2 1 0:23

Autogamous cells from the indicated crosses were isolated and later tested for their ability to produce serotype A. The ratios of A producers (A') to nonproducers (A-) are given in the table. The cytoplasmic parent of each group of F2 progeny is indicated for each exconjugant.

run onthese A- F2 cultures onewould expect that the half of these clones of genotype A-'300/A"300 would have the - 1300 deletion. The otherhalf of theclones of genotype A5'/A5' that are derived from the d48 exconjugant in which the old macronucleus contains a +1 deficiency, would be expected to have the +1 deficiency. These predictions were confirmed by PCR; four F2 clones derived from a d48 exconjugant were found to be +1 and four were -1 300. It is more difficult to predict what might be the nature of the deletion in the A- A5'/As' segregants derived from the d12(-1300)exconjugant.In this case the old macronucleus that influences the nature of the newly forming macronuclei has a -1300 deletion and not a +1 deletion. Similar PCR tests were run on F2 segregants derived from asingle d12(-1300) exconjugant, all of which were A-. Of 23 F2 clones 7 were +1 and 16 were -1 300 deletions. Although the fit to a 1 :1 ratio is far from perfect in this case, a chi square test shows that the fit is not significantly different from 1:l (chi square with Yates correction = 2.78, probability 0.1). Thus, the A- A5'/A5' segregants from the d 12 exconjugant had the + 1 deletionand theposition of the deletion was not affected by the -1 300 allele

FIGURE4.-I'CR analysis of I;? segregation. A pair of primers spanning the region from - 136 1 to -5 12 was used with 100 cells inPCRin order to distinguish d12(+1) molecules which give an 850-bp PCR product from d l 2(-1300) molecules which give no product. A portion of each reaction product was run on an agarose gel and stained with ethidium bromide. Lane 1, dl 2(-1300). Lane 2, d l 2(+1). Lanes 3-6, four of the A- FYsegregant clones obtained by autogamy from a cross of d 12(- 1300) X 5 1 show no band and hence are d 12(- 1300). Lanes 7-1 0 , four randomly chosen F2clones from the cross d 12(- 1300) X d 12(+ 1) (all clones from the cross were A-.) Lanes 8 and 10 show no band and hence are d 12(- 1300): lanes 7 and 9 show the product characteristic of d12(+1): M. markers. A PCR product of the predicted size, 850 bp. is found only in d 12(+1) and not in d 12(- 1 300). TABLE 2 Tests for allelism: Fnratios from exconjugants of d4-6 X d l 2 crosses

Cross

d4-6A'

X

Progeny of d4-6 exconjugant

d I2(+ 1)A-

d4-6A+ X d 12(-1300 1)A-

A':A-

Progeny of d l 2 exconjugant A+:A-

11:16 15:21 10:13 14:10 9:11 Sum = 5 9 7 1 14:27 7:17 8:12 14:U 10:12 149 9:15 Sum = 76: 100

Sum for both crosses = 135: 17 1 Autogamous cells from the indicated crosses were isolated and later tested for their ability to produce serotype A. d4-6 homozygous for the allele A'". The ratios of A producers (A') to nonproducers (A-) are given in the table. The cytoplasmic parent of each group of F2 progeny is indicated for each exconjugant. All lines were 29A.

in the old macronucleus. We conclude that the deficiencies in wild-type A alleles, produced innewly forming macronuclei by a lack of wild-type A genes in the old macronuclei, are deficiencies that start at 1,

+

Affecting Mutants TABLE 3

Fnratios from crosses of KOIZUMI’Smicronuclear transplant strains Progeny of 5 1 exconjugant Cross

A+:A-

51A+ X K1-2A-

47:O 19:19 48:O

5 1A+X K2-2A-

23:O 51Af

X

41:O 48:O

K4-1 23: 1 24:O 24:O

Progeny of KOIZUMI exconjugant A+:A-

0:42 40:5

0:46 0:35 0:32 5:19 24:O

Autogamous cells from the indicated crosses were isolated and later tested for their ability to produce serotype A. The ratios of A producers (A+) to nonproducers (A-) are given in the table. The cytoplasmic parent of each group of F2 progeny is indicated for each exconjugant.

even when the old macronucleus is derived from d l 2 with a deficiency starting at - 1300. Finally, an apparenteffect of micronucleoplasm on the non-Mendelian determination is seen in the d l 2 X d48 crosses. In these crosses the d l 2 exconjugants often produce A- FI clones and give an excess of Aat thenext autogamy. Howeverin the cross d l 2 x 5 1 the heterozygous d 12 exconjugantalways changes to A+ in the F1 and gives a 1 :1 ratio of A+:A- in the F2 by autogamy. Thus a wild-type A” allele received from a5 1 wild-type cell is lesslikely to suffer deletions than a wild-type A5’ allele received from a d48 cell. Creation of pseudod48 lines: If our idea about the nature of d48 is correct, it shouldbe possible to recreate ad48 strain artificially by micronuclear transplantation. KOBAYASHIand KOIZUMI(1 990)removed the micronuclei from several individuals of d 12 (starting point of the dl2 deletion notknown) and replaced them with micronuclei from 51 wild type. Since such cells have macronuclei with few copies of the A gene, and micronuclei with normal A genes, they should be incapable of producing normal macronuclei with intact A genes at autogamy. In fact, they shouldbe exactly like d48, even though derived from wild type andd12.They should breedtrue as A- strains through vegetative divisions and also through autogamy. Furthermore, they should behave like d48 in crosses. The data given in Table 3 representthe results of crosses between wild-type and the Koizumi strains. They are typical of the results obtained when wild type is crossed to d48 (EPSTEINandFORNEY 1984), most pairs yielding A+ clones from the wildtype exconjugant and A- from the transplant exconjugant, both in F1 and F P . T h e occasional A+ clones arising from the A- transplant exconjugant are also typical of d48. In two cases A- lines were derived from a wild type exconjugant (19 A+:19 A- and 23 A+:l A-. See Table 3). This result was unexpected,

DNA Processing

53

and nofollow-up was made in these cases to investigate the nature of the A- lines. Tests using PCR showed that all the chimeric lines contained +1 deletions. A cross of one of the Koizumi “pseudo d48”strains to d l 2(+1) (data not shown) is consistent with the results obtained by KOBAYASHI and KOIZUMI (1 990) in a similar cross. T h e phenotypes of the progeny were similar to those obtained in the cross of d48 x d12(+1). See Figure 7. Downstream A gene sequences are present in dl2 micronuclei andin low copy number in micronuclei: Perhaps the simplest conclusion concerning the two d l 2 deletions is that the micronuclei have sustained deletions which are passed on to themacronuclei. On the other hand, data shown below suggest that the micronuclei of the d 12 strains contain possibly comdeletions occur plete, but mutantA genes, and that the during the formation of new macronuclei at conjugation and autogamy. T o probe directly for the presence of the downstream A gene sequences in d12, PCR reactions were carried out. T w o 20-bp primers were constructed for aregion that is about 5kbdownstreamfrom the apparent break points for macronuclear DNA in d48 and d12. The primers direct amplification of a 395bpregionstarting at position 5107andendingat 5501.SeeFigure 1. T h e region also contains an asymmetrically placed Hind111 site at 5277, which is useful in identifying the amplified product. Figure 5 shows the results of PCR on whole cell DNA from 5 1, d48 and d12.All three DNA preparations contain the downstream region. In each case, Hind111 digestion yields two fragments of the expected sizes (222 bp and173 bp),confirmingthe identification of the product. T o obtain a rough estimate of the relative number of the putative full length A genes in d l 2, d48 and 5 1, we carried out PCR reactions with fixed numbers of whole cells as starting material. The results are shown in Figure 6. Positions are displaced in some cases because of overloading. T h e results suggest that the number of copies of the downstream region in d48 is considerably less than the number in wild-type strain 51, yet more than the number in d12. If this were the case, it would indicate that d48 contains a small number of A genes in its macronucleus. T o investigate this possibility we obtained clones of amicronucleate d48 and d12. PCR carried o u t on the amicronucleate d48 and d l 2 both showed that the macronuclei do indeed contain the sequences, but in low copy number (data notshown). Thus, the defects in DNA processing are not absolute, and a few copies of possibly intact A genes are present in macronuclei of all the mutants. We conclude that full length A gene molecules are probably present in both themicronuclei and macronuclei of all the mutants, butin very low copy number

B. Rudman et al.

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FIGURE5.-PCR detection of d~wnstreamA gene sequences in the mutants. A pair of primers spanning the region from 5107 to 550 I was used with 1 pg of purified DNA in PCR in order todetect the presence of downstream A gene sequences. PCR products were purified and portions cut with HindIII. The HindIII site in the 395bp region of the A gene spanned by the primers starts 222bp from one end of the region and 173 from the other end. Appropriate quantities of the uncleaved and cleaved DNAs (lanes designated U and C, respectively)were run on a 5% acrylamide gel. M, markers. Lanes 1 and 2, d48. Lanes 3 and 4, d12(-1300). Lanes 5 and 6, 51. Lanes 7 and 8, pSA14SB. All the mutants appear to have the downstream sequences. Control uncut DNAs in lane 9 (from the unrelated ciliate, Colpidium) and lane 10 (from Escherichia coli) fail to show the 395-bp PCR product.

in the macronucleus. Truncated A genes make up the bulk of the macronuclear copies. DISCUSSION

Nature of the Mendelian mutations:T h e d 12 micronuclear alleles A-""" and A+' always give rise to macronuclear deletions beginning at -1 300 and +1 respectively, even when they are present in heterozygous combinations with each other or with wild-type alleles. Thus, in a cross of d l 2(-1300) to wild type, both F1 exconjugants, even though they are A+, each contains -1300 as well as wild-type DNA molecules in the macronucleus. Moreover, deletions associated with the A"300 and A+' alleles are not influenced by the composition of the old macronucleus during formation of new macronuclei; they always produce deletions. On the basis of the genetic behavior alone, one might concludethat these two alleles represent simple micronuclear deletions. However, certain observationssuggest that thed 12 phenotype is not due to micronuclear deletion. First, stock in two instances we havefound A+cellsin cultures of d 12. Barring the unlikely chance of contamination in two separate cases, these represent reversions of d l 2 to wild type. Second,strains with extensive deletions such as d l 2(-1300) and d 1(- 1000) havegiven rise to strains with lessextensive and dl(+ 1). Third, PCR analydeletions, e.g., d 12(+ 1)

FIGURE6.-Effectiveness of different numbers of cells as templates for PCR. Cells of d48, 5 I and d 12 were used as templates for primers spanning the region 5 10 1 to 5495. Lanes 1, 2, 3, d48: 10, 60, 600 cells, respectively. Lanes 4, 5, 6, 5 1:1, 10, 60 cells, respectively. Lanes 7, 8, 9, d12: 10, 60,600 cells, respectively. The results suggest that d48 has fewer copies of A gene than wild type, and that the amount in d 12 is even less.

sis showed that the micronuclei in the d l 2 lines contain downstream regions of the A genes as well as the upstream flanking portion. PCR analysis on amicronucleate d l 2 lines indicated that a small number of possibly intact A genes are present in the macronuclei as well. These facts argue against the view that the mutants represent simple micronuclear deletions. We conclude that A"300 and A+' are processing mutants, i e . , modified micronuclear A genes that yield deletions at specified regions when the micronuclei produce macronuclei at autogamy and conjugation. However, final proof requires isolation and characterization of the micronuclear A gene from d12. Alleles that affectserotype specificity, as well as alleles affecting other traits, are normally extremely stable in Paramecium. In spite of intensive investigations therearenoreports,toour knowledge, of changes in specificity alleles. However, there are numerous reportsof changes in the alleles that affect the position of the macronuclear A gene deletions, such as the d l 2 alleles. This lability evidently also applies to d l and to the d l 6 mutant described by EPSTEIN and FORNEY(1984). d 16 was isolated in a screen for A- mutants, hence presumably was defective at the A locus when first isolated. Later, however, the deletion point was found to be justdownstream of the 3' end of the A gene, and the capacity to produce serotype A was then identical to that of wild type. Whether these frequent changes can be accounted for by mobile elements or by some other means will have to await molecular characterization of the micronuclear copies of the various alleles of the A gene. Non-Mendelian phenomenon: properDNA processing requiresmacronuclear A genes: T h e nonMendelian phenomenon consists of a failure of A''

DNA Mutants Affecting

micronuclear alleles toundergoproper processing because of macronuclear deficiencies in d48 or d12. Direct evidence for the sensitivity of the wild-type A gene processing to the macronuclear environment is provided by the observation that new lines with all the properties of d48 may be artificially constructed from d l 2 bysimply replacing its micronuclei with micronuclei from 51 wild-typecells (KOIZUMI and KOBAYASHI1990). This improper processing yieldsdeletions in the A'' gene sometimes, but not always. A number of factors can be identified which influence the probability of occurrence of deletions. First, The major factor is whether the old macronucleus in the cell in which autogamy or conjugation is taking place contains a downstream region of the A gene ( Y o u et al. 1991; unpublished work in our laboratory). If this region is present in sufficiently high numbers, as it is in wild type, then the deletions never occur. If the region is not present in high numbers, as in the case in d48, the deletions usually occur. No satisfactoryhypothesishasbeensuggested to account for this effect. KOIZUMI and KOBAYASHI ( 1989) provided evidence that 5 1 wild-type cytoplasm could rescue d48, provided it is taken from autogamous cells and injected into autogamous cells. However,no smaller transcripts from the A gene that might encode a cytoplasmic factor have beendetected (H. JESSOP-MURRAY, unpublished results). Perhaps portions of the A genes themselves may be liberated from the old degenerating macronucleus and play a direct role inDNA processing. Alternatively, they may act to bind a DNA processingfactor to which the newly forming wild-type A genes are sensitive, thereby sequestering the processing factor and preventing it from making aberrant cleavages. However, the assumption that cytoplasmicDNAmolecules bring about rescue does not fit wellwith the finding of KOIZUMIand KOBAYASHI(1989) that injection of the cloned A gene into autogamous d48 cells gives only low level rescue. Whatever the mechanism ofprotection provided by the A genes in the old macronucleus, it is clear that when it fails because ofdeletions starting at either 1 or -1300 in the old macronucleus, the new macronucleus shows only the deletions starting at + l . This indicates that the sectionofDNA extending from -1300 to +1, which is present in 1 macronuclei and missingin -1300macronuclei,has no role in the determination of the deletion site. This result agrees with the finding in our laboratory (H. JESSOP-MURRAY, unpublished results) that this region is not able to prevent breaks and effect rescue of d48. Only a downstream portion of the A gene is effective in this way. Second, a major exception to the dependence of proper processing on the presence of downstream A

+

+

55

Processing W

x

d4a

WXd12

d40 X d l 2

P

FER

MAC DEV I

I

FIGURE7.-A model to explain and summarize the results of crosses. The two kinds of d l 2 give similar results in crosses. It is assumed that a downstream portion of the wild-type A allele and the mutant alleles, A+' and A"3"" (designated in the figure as a ) produce a factor necessary for proper processing. Production of the factor occurs only during conjugation and autogamy. The A genes do not produce the factorin the micronuclei and only rarely produce it in macronuclear anlagen (explaining the rare production of A+ clones from both exconjugants in the cross of d48 X 51). Production of the factor regularly takes place in macronuclear fragments. In wild type the factor passes through the cytoplasm and into the macronuclear anlagen. By contrast, in d48 the macronuclear fragments lack the A gene and produce no factor. Therefore A genes are usually not included in the new macronuclei forming in d48 cellswhen d48 conjugates withwild type. On the other hand, the mutant alleles in d l 2 usually produce the factor in early anlagen of heterozygotes and normal processing of A genes occurs. Since the mutant d l 2 alleles cannot respond to the factor, most are deleted from macronuclei before macronuclear formation is completed. Nothing is known aboutthe nature of the factor. For example, it is possible that the alleles themselves may act as the factor.

gene sequences in the old macronucleus is provided by the behavior of the wild-type A'' allele when it is in heterozygous combination with the A+' or A"300 d l 2 alleles.Wild-type micronuclear alleles in such heterozygotes are often properly processed in spite of a reduced number of A genes in the old macronucleus. It is surprising that a mutation that causes defective processinghas a "protective" effectonwild-type genes. Third, at conjugation, migratory gametic micronucleiwithidentical genotype but derived from different lines, may differ in their effect onthe frequency of deletions. We have observed that while d48 is very stable in the cross d48 X d48, in the cross d48 X 5 1,

56

B. Rudman et al.

the d48 exconjugant changes quite frequently to A+. Thus, a migratory gametic nucleus from 5 1 wild type yields more intact chromosomes ind48 than a migratory gametic nucleus from d48, eventhoughboth nuclei contain the wild type A’’ allele. Furthermore, in the cross d l 2 X 5 1, the heterozygous d l 2 exconjugant always changes to A+ in the F1 and gives a 1:l ratio of A+:A- in the F2 by autogamy. In the d l 2 X d48 cross, however, the d l 2 exconjugants, which are supposedly identical with the d l 2 exconjugants of the d l 2 X 5 1cross, often produce A- F1 clones and give an excess of A- progeny at the next autogamy. In both these examples, the A5‘ wild-type allele received froma51 wild-type cell islesslikely toundergo deletions than a A” wild-type allele received from a d48 cell. These differences do not appear to be due to the transfer of cytoplasm between conjugants that occurs when cytoplasmic bridges areproduced by “delayed separation” (SONNEBORN 1975), although the possibility that minute amounts of cytoplasm are exchanged cannot be eliminated. Another possibility is that the micronucleoplasm derived from 5 1 wild-type cells acts much like the macronucleoplasm from 51 wild-type cells to prevent deletions in developing macronuclei. A similar effect was demonstrated in the case of mating type inheritance in P. tetraurelia (BRYGOO et al. 1980). Theyused crosses of different mating types to amicronucleate lines to demonstrate the effect. In view of these considerations, it is probably more accurate to conclude that the non-Mendelian phenomenon is due to effects of both micronucleoplasm and macronucleoplasm, rather than simply macronucleoplasm. Figure ’7 represents an attempt to provide a summary of the results of the crosses and a possible explanation as detailed in the figurelegend. It should be emphasized that this working model is only one of several possible explanations. Related phenomena: T h e involvement of the old macronucleus in the formation of the new macronucleus at autogamy and conjugation is likely to be of general occurrence and notsimply restricted to a few specialized cases like the A gene. The inheritance of mating type in P. tetraurelia has been extensively 1975) and theeffect studied (reviewed in SONNEBORN of the old macronucleus on the new macronucleus in mating type determination iswell established. Even the mating type “selfers” have their parallel in the “mixed” clones (see MATERIALS AND METHODS) which contain both wild type and d48 cells. Similar systems of inheritance appear to exist in the case of the trichocyst mutants in P . tetraurelia (SONNEBORNand SCHNELLER 1979) and in a case involving surface proteins in Tetrahymenathermophila (DOERDERand BERKOWITZ 1987).Moreover,preliminaryexperi-

ments (our unpublished results) on new mutants unable to express surface proteins B and D also seem to show non-Mendelian as well as Mendelian inheritance. Since all the deletions of the A gene remove the entire end of the macronuclear chromosome bearing the A gene,prerequisitesfor thephenomenon may be a location adjacent to nonessential downstream sequences and proximity tothe telomere. Deletions might otherwise prove to be lethal. We thank GUY HAMILTON technical for assistance and JAMFS D. FORNEY for theoriginal cultures of mutants d l and d l 2. This work was supported by U.S. Public Health Service grant GM 31745 from the National Institutes of Health.

LITERATURE CITED BRYGOO, V., T . M. SONNEBORN, A. M. KELLER, R. V. DIPPELLand M. V. SCHNELLER, 1980 Genetic analysis of mating type differentiation in Paramecium tetraurelia. 11. Role of the micronucleus in mating type determination. Genetics 94: :951 -959. DOERDER, F. P., and M. S. BERKOWITZ, 1987 Nucleo-cytoplasmic interaction during macronuclear differentiation in ciliate protists: genetic basis for cytoplasmic control of SerH expression during macronuclear development in Tetrahymena thermophila. 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: 1583-1 590. FORNEY,J. D., and E. H. BLACKBURN, 1988 Developmentally controlled telomere addition in wild-type and mutant paramecia. Mol. Cell. Biol. 8: 251-258. FORNEY, J. D., L. M. EPSTEIN,L. B. PREER,B. M. RUDMAN, D. J. WIDMAYER, W. H. KLEINandJ. R. PREER, JR., 1983 Structure and expression of genes for surface proteins in Paramecium. Mol. Cell. Biol. 3: 466-474. GODISKA,R., K. J. AUFDERHEIDE,D. GILLEY,P. HENDRIE, T. FITZWATER, L. PREER,B. POLLSKY 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. KOBAYASHI,S., and K. KOIZUMI, 1990 Characterization of nonMendelian and Mendelian mutant strains by micronuclear transplantation in Parameciumtetraurelia. J. Protozool. 37: 489-492.

KOIZUMI, S., and S. KOBAYASHI, 1989 Microinjection ofplasmid DNA encoding the A surface antigen of Paramecium tetraurelia restores the ability to regenerate a wild-type macronucleus. Mol. Cell. Biol. 9 4398-4401. SONNEBORN, T. M., 1950 Methods in the general biology and genetics of P. aurelia.J. Exp. Zool. 113: 87-143. SONNEBORN, T. M., 1975 Parameciumaurelia, pp. 469-594 in Handbook of Genetics, Vol. 2, edited by R. KING. Plenum Press, New York. SONNEBORN, T. M., and M. V. SCHNELLER, 1979 A genetic system for alternative stable characteristics ingenomically 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