Analysis of the micronuclear B type surface protein gene in Paramecium tetraurelia.

The micronuclear DNA of Paramecium contains sequences that are precisely excised during the formation of the macronuclear (somatic) genome. In this paper we show that four eliminated sequences ranging in size from 28 to 416 base pairs, are present in or near the micronuclear copy of the B surface protein gene. Each excised sequence is bounded by the dinucleotide 5'-TdA-3'. Comparison of the micronuclear B gene with the previously determined micronuclear sequence of the A surface protein gene shows that although the positions of at least three of the eliminated sequences are conserved in both genes, the sequences are highly divergent. Transformation of vegetative macronuclei with fragments of the micronuclear B gene results in replication and maintenance of the DNA, but the micronuclear specific sequences are not removed. Previous studies have shown that the correct incorporation of the B gene into the new macronucleus requires copies of the macronuclear B gene in the old macronucleus. Using macronuclear transformation, we show that the micronuclear B gene can substitute for the macronuclear B gene with regard to its role in DNA processing. This suggests that the macronuclear DNA is not acting as a guide for the excision of the micronuclear specific sequences.     

A Mendelian Mutation Affecting Mating-Type Determination Also Affects Developmental Genomic Rearrangements in Paramecium Tetraurelia

In Paramecium tetraurelia, mating type is determined during the differentiation of the somatic macronucleus from a zygotic nucleus genetically competent for both types, O and E. Determination of the developing macronucleus is controlled by the parental macronucleus through an unknown mechanism resulting in the maternal inheritance of mating types. The pleiotropic mutation mtF(E) affects macronuclear differentiation. Determination for E is constitutive in mutant homozygotes; a number of unrelated mutant characters are also acquired during development. We have examined the possibility that the mutation causes a defect in the developmental rearrangements of the germ-line genome. We show that the excision of an IES (internal eliminated sequence) interrupting the coding sequence of a surface antigen gene is impaired in the mutant, resulting in an alternative macronuclear version of the gene. Once established, the excision defect is indefinitely transmitted across sexual generations in the cytoplasmic lineage, even in a wild-type genetic context. Thus, the processes of mating-type determination and excision of this IES, in addition to their common sensitivity to the mtF(E) mutation, show a similar maternal inheritance of developmental alternatives in wild-type cells, suggesting a molecular model for mating-type determination.     

Sequence-specific epigenetic effects of the maternal somatic genome on developmental rearrangements of the zygotic genome in Paramecium primaurelia.

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences.     

Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.     

Trichocyst phenotype transformation induced by macronuclear transplantation in Paramecium tetraurelia

A portion of the macronucleus of wild-type cells of Paramecium tetraurelia was removed and was injected into cells homozygous for the ftA mutation. The ftA mutants make defective trichocysts and are unable to perform normal trichocyst exocytosis. After injection, approx. 30% of the surviving cells show a phenotype shift from mutant to wild-type. This shift is stable during subsequent vegetative growth until clonal death. If, however, the hybrid cell lines are brought to autogamy (which discards the existing macronucleus and forms a new one from sexual products derived from a micronucleus), then the lines revert to the ftA phenotype. Since micronuclei were not transplanted, the phenotypic reversion after autogamy is to be expected, and demonstrates that the transformation affects the macronucleus only. A second series of injections involved transfer of a portion of the macronucleus from cells homozygous for the trichocyst ptA mutation into ftA host cells. These two mutations are genetically complementary, so the injection should be genetically equivalent to forming a double heterozygote. Approx. 20% of the injection survivors shift to wild-type. This shift is also vegetatively stable unless autogamy occurs; after autogamy, reversion to the ftA phenotype is seen. These results show that a portion of a macronucleus can be successfully transplanted from one cell to another and that, in the host cytoplasmic environment, normal gene expression and replication of a transplanted macronucleus does occur. The technique of macronuclear transplantation is significant to studies of the macronuclear contribution to clonal aging, and to studies on genetic control over trichocyst development.     

Developmentally controlled telomere addition in wild-type and mutant paramecia.

We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200- to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition.     

Molecular Biology of the Genes for Immobilization Antigens in Paramecium1

Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition lo known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51Cand 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.     

The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed. © 1992 Wiley-Liss, Inc.     

Nucleo-cytoplasmic interaction during macronuclear differentiation in ciliate protists: genetic basis for cytoplasmic control of SerH expression during macronuclear development in Tetrahymena thermophila

A novel class of mutations affecting the developmental expression of SerH cell surface antigen genes of Tetrahymena thermophila is described. Unlike previous categories of mutation, the four independently isolated mutations of this class act through the cytoplasm to affect SerH genes during macronuclear development. That is, macronuclei which develop under the influence of mutant cytoplasm do not subsequently express H, most likely because the developmental processing of SerH genes is affected. The cytoplasmic effect is specific for the SerH locus and is independent of which SerH allele is present. In place of H, hitherto unknown antigens are expressed. Expression of SerH can be rescued during development either by wild-type cytoplasm exchanged between conjugants or by the homozygous wild-type genotype. The mutations segregate independently of the SerH genes and identify one, possibly two, bistable genes. Possible models to explain these results are discussed.     

Molecular biology of the genes for immobilization antigens in Paramecium

Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition to known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51C and 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.     

RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia

The germ line genome of ciliates is extensively rearranged during development of the somatic macronucleus. Numerous sequences are eliminated, while others are amplified to a high ploidy level. In the Paramecium aurelia group of species, transformation of the maternal macronucleus with transgenes at high copy numbers can induce the deletion of homologous genes in sexual progeny, when a new macronucleus develops from the wild-type germ line. We show that this trans-nuclear effect correlates with homology-dependent silencing of maternal genes before autogamy and with the accumulation of approximately 22- to 23-nucleotide (nt) RNA molecules. The same effects are induced by feeding cells before meiosis with bacteria containing double-stranded RNA, suggesting that small interfering RNA-like molecules can target deletions. Furthermore, experimentally induced macronuclear deletions are spontaneously reproduced in subsequent sexual generations, and reintroduction of the missing gene into the variant macronucleus restores developmental amplification in sexual progeny. We discuss the possible roles of the approximately 22- to 23-nt RNAs in the targeting of deletions and the implications for the RNA-mediated genome-scanning process that is thought to determine developmentally regulated rearrangements in ciliates.     

The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement.

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed.     

A mutation in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements.

The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28-882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tcl transposons (KLOBUTCHER and HERRICK 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES259 I has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its Mild-type micronuclear copy through multiple sexual generations.     

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

Removal of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5-azacytidine, 5-aza-2'- deoxycytidine and 5-fluoro-2'-deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6-azacytidine, 2'-fluoro-2'-deoxy-cytidine and cytosine-β-D-arabinofuranoside did not. These results suggest that (i) 5-methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.