The intranuclear organization of normal, hemizygous and excision-deficient rRNA genes during developmental amplification in Tetrahymena thermophila

In the ciliated protozoan, Tetrahymena thermophila, the diploid germinal micronucleus contains two allelic copies of the gene for ribosomal RNA (rDNA). During genesis of new somatic macronuclei the germline rDNA gene is excised by developmentally programmed chromosome breakage and preferentially amplified to ∼9,000 copies. We have studied this process by fluorescence in situ hybridization. We find that initially rDNA amplification is restricted to two separate and highly confined regions of the nucleus. Analysis of nuclei that are hemizygous for the rDNA locus reveals that each focus of hybridization is derived from a single allele of the rDNA. As rDNA amplification progresses these two foci of hybridization disperse and spread throughout the macronucleus, eventually forming ∼100–500 new nucleoli. These events are correlated with morphologically distinct developmental stages. We investigated the amplification of the C3 allele of the rDNA that confers a replication advantage over the B allele during vegetative propagation, and find no evidence for preferential amplification of the C3 early in rDNA maturation. We also show that the rmm 11 rDNA mutant allele, which is defective for developmentally programmed rDNA excision, can be amplified during the two-foci stage in mutant homozygotes and heterozygotes, but fails to amplify further and disperse into multiple nucleoli. These data indicate that amplification of the rmm 11 allele is not delayed during the initial rounds of amplification, and suggest that efficient excision is not required for this amplification to occur. We propose that rDNA amplification is a two-step process. First, the two rDNA alleles are independently amplified, while allelic copies remain closely associated. Later, copies of the rDNA disperse and are further amplified, presumably because rDNA excision has occurred, generating fully mature rDNA minichromosomes that are able to replicate to high copy number. Received: 21 February 1997; in revised form: 21 April 1997 / Accepted: 5 May 1997     

A yeast mutation that stabilizes a plasmid bearing a mutated ARS1 element.

To identify the trans-acting factors involved in autonomously replicating sequence (ARS) function, we initiated a screen for Saccharomyces cerevisiae mutants capable of stabilizing a plasmid that contains a defective ARS element. The amm (altered minichromosome maintenance) mutations recovered in this screen defined at least four complementation groups. amm1, a mutation that has been studied in detail, gave rise to a 17-fold stabilization of one defective ARS1 plasmid over the level seen in wild-type cells. The mutation also affected the stability of at least one plasmid bearing a wild-type ARS element. amm1 is an allele of the previously identified TUP1 gene and exhibited the same pleiotropic phenotypes as other tup1 mutants. Plasmid maintenance was also affected in strains bearing a TUP1 gene disruption. Like the amm1 mutant, the tup1 disruption mutant exhibited ARS-specific plasmid stabilization; however, the ARS specificities of these two mutants differed. The recovery of second-site mutations that suppressed many of the tup1 phenotypes but not the increased plasmid maintenance demonstrates that the plasmid stability phenotype of tup1 mutants is not a consequence of the other defects caused by tup1.     

Alteration of the Tetrahymena Genome During Nuclear Differentiation*†

Synopsis.
The DNA of the macro- and the micronucleus of Tetrahymena thermophila has been compared by various biochemical methods. It became evident from their thermal denaturation temperatures and buoyant densities that the 2 DNAs were very similar in overall composition. Small differences were detected when the sequence complexities of these DNAs were compared by DNA renaturation studies. The studies suggested that ˜ 10% of the micronuclear genome was lost or underrepresented in the macronucleus. Comparison of individual gene levels revealed further differences. By using the technic of gene cloning a micronuclear sequence was isolated which hybridized only with micronuclear, but not with macronuclear DNA. These results indicated the occurrence of elimination or underreplication of this sequence in the macronucleus. Gene amplification was also shown to occur. In the micronucleus only a single copy of rDNA was found integrated into the chromosome. During macro-nuclear development, amplification was observed to occur, and the amount of rDNA to increase, until there were ˜ 200 copies per haploid genome in the mature macronucleus. all of them extrachromosomal and palindromic. The 3rd case of alteration involved a simple repeated sequence, (CCCCAA)n, present in the termini of rDNA and also in many other locations of the genome. Restriction endonuclease digestion studies revealed drastic differences in the organization of the repeats between macro-and micronucleus. These differences may be interpreted as the results of chromosome fragmentation which occurs at every cluster of the repeats during macronuclear development. The relationship between this event and gene amplification and elimination is discussed.     

Control of rDNA replication in Tetrahymena involves a cis-acting upstream repeat of a promoter element

A novel genetic scheme was used to isolate mutants altered in the formation or maintenance of amplified rDNA in the Tetrahymena macronucleus. One such mutant had a cis-acting rDNA mutation that affected the ability of mutant rDNA molecules to replicate in macronuclei in the presence of a wild-type (B strain) rDNA. The mutant rDNA was lost from these heterozygous macronuclei during vegetative cell divisions, although it was maintained normally in the homozygous or hemizygous state. In contrast, wild-type macronuclear rDNA of the C3 strain used to obtain the mutant outreplicated B strain rDNA in B/C3 heterozygote macronuclei. Sequence differences were found between wild-type B and C3 and mutant C3 rDNAs in the replication origin region, changing an upstream repeat of a highly conserved rRNA promoter element. We propose that the various rDNA alleles differentially compete for limiting amounts of trans-acting factors that bind to these enhancer-like repeats and positively regulate rDNA replication.     

Evolutionary Conservation of Sequences Directing Chromosome Breakage and Rdna Palindrome Formation in Tetrahymenine Ciliates

Extensive, programmed chromosome breakage occurs during formation of the somatic macronucleus of ciliated protozoa. The cis-acting signal directing breakage has been most rigorously defined in Tetrahymena thermophila, where it consists of a 15-bp DNA sequence known as Cbs, for chromosome breakage sequence. We have identified sequences identical or nearly identical to the T. thermophila Cbs at sites of breakage flanking the germline micronuclear rDNA locus of six additional species of Tetrahymena as well as members of two related genera. Other general features of the breakage site are also conserved, but surprisingly, the orientation and number of copies of Cbs are not always conserved, suggesting the occurrence of germline rearrangement events over evolutionary time. At one end of the T. thermophila micronuclear rDNA locus, a pair of short inverted repeats adjacent to Cbs directs the formation of a giant palindromic molecule. We have examined the corresponding sequences from two other Tetrahymena species. We find the sequence to be partially conserved, as previously implied from analysis of macronuclear rDNA, but of variable length and organization.     

Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.     

The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila.

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia.     

Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA.

Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development. In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction. The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development. The breakage event is associated with DNA elimination and telomeric sequence addition. Similar results were also found previously for the 3' junction of this gene. These results could explain how the extrachromosomal rDNA is first generated. Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found. These sequences might serve as signals for site-specific breakage. In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene. The repeats are 42 nucleotides long and are separated by 28 nucleotides. The existence of this structure provides a simple explanation for the formation of the palindromic rDNA.     

Mutants of S. CEREVISIAE Defective in the Maintenance of Minichromosomes

We have isolated yeast mutants that are defective in the maintenance of circular minichromosomes. The minichromosomes are mitotically stable plasmids, each of which contains a different ARS (autonomously replicating sequence), a centrometeric sequence, CEN5, and two yeast genes, LEU2 and URA3. Forty minichromosome maintenance-defective (Mcm-) mutants were characterized. They constitute 16 complementation groups. These mutants can be divided into two classes, specific and nonspecific, by their differential ability to maintain minichromosomes with different ARSs. The specific class of mutants is defective only in the maintenance of minichromosomes that carry a particular group of ARSs irrespective of the centromeric sequence present. The nonspecific class of mutants is defective in the maintenance of all minichromosomes tested irrespective of the ARS or centromeric sequence present. The specific class may include mutants that do not initiate DNA replication effectively at specific ARSs present on the minichromosomes; the nonspecific class may include mutants that are affected in the segregation and/or replication of circular plasmids in general.     

Synthesis of ribosomal DNA in conjugating Tetrahymena.

In the ciliate protozoan Tetrahymena thermophila, a single integrated gene coding for ribosomal RNA in the micronucleus is amplified during the sexual cycle to yield many copies of extrachromosomal palindromic rDNA in the macronucleus. Hybridization of newly synthesized DNA with rRNA has shown that extensive rDNA synthesis takes place early in the sexual cycle of Tetrahymena. The number of genes synthesized during this period is sufficient to account for gene amplification. A later period of rDNA synthesis occurs when new macronuclear anlagen are beginning to develop. This synthesis may represent preferential polyploidization of already amplified rDNA.     

TIF1 Represses rDNA replication initiation, but promotes normal S phase progression and chromosome transmission in Tetrahymena

The non-ORC protein, TIF1, recognizes sequences in the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome that are required for origin activation. We show here that TIF1 represses rDNA origin firing, but is required for proper macronuclear S phase progression and division. TIF1 mutants exhibit an elongated macronuclear S phase and diminished rate of DNA replication. Despite this, replication of the rDNA minichromosome initiates precociously. Because rDNA copy number is unaffected in the polyploid macronucleus, mechanisms that prevent reinitiation appear intact. Although mutants exit macronuclear S with a wild-type DNA content, division of the amitotic macronucleus is both delayed and abnormal. Nuclear defects are also observed in the diploid mitotic micronucleus, as TIF1 mutants lose a significant fraction of their micronuclear DNA. Hence, TIF1 is required for the propagation and subsequent transmission of germline chromosomes. The broad phenotypes associated with a TIF1-deficiency suggest that this origin binding protein is required globally for the proper execution and/or monitoring of key chromosomal events during S phase and possibly at later stages of the cell cycle. We propose that micro- and macronuclear defects result from exiting the respective nuclear S phases with physically compromised chromosomes.     

Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila.

We have investigated the occurrence of methylated adenine residues in the macronuclear ribosomal RNA genes of Tetrahymena thermophila. It has been shown previously that macronuclear DNA, including the palindromic ribosomal RNA genes (rDNA), of Tetrahymena thermophila contains the modified base N-6-methyladenine, but no 5-methylcytosine. Purified rDNA was digested with restriction enzymes Sau 3AI, MboI and DpnI to map the positions and levels of N-6-methyladenine in the sequence 5' GATC 3'. A specific pattern of doubly methylated GATC sequences was found; hemimethylated sites were not detected. The patterns and levels of methylation of these sites did not change significantly in different physiological states. A molecular form of the rDNA found in the newly developing macronucleus and for several generations following the sexual process, conjugation, contained no detectably methylated GATC sites. However, both the bulk macronuclear DNA and palindromic rDNA from the same macronuclei were methylated. Possible roles for N-6-methyladenine in macronuclear DNA are discussed in light of these findings.     

Ribosomal RNA gene amplification in Tetrahymena may be associated with chromosome breakage and DNA elimination

The chromosomal DNA sequence adjacent to one end of the single ribosomal RNA gene (rDNA) in the micronucleus of Tetrahymena has been isolated by cloning. Using this sequence as a hybridization probe the organization of the same sequence in the somatic macronucleus has been examined. The restriction enzyme digestion maps of this sequence in the two nuclei are very different. Detailed mapping studies suggest that a chromosome break has occurred near the junction between the rDNA and the neighboring sequence during the formation of the macronucleus. As a result the flanking sequence is located near a free chromosome end in the macronucleus. The existence of such a linear DNA end has also been shown by digestion with the exonuclease Bal 31. In addition to the breakage, some sequences at this junction are found to be eliminated from the macronucleus. This observation has been interpreted in relation to the mechanism of rDNA amplification, which in Tetrahymena generates extrachromosomal rDNA molecules during macronucleus development.     

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.     

migS, a cis-acting site that affects bipolar positioning of oriC on the Escherichia coli chromosome

During replication of the Escherichia coli chromosome, the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for bipolar migration is located in this region. To identify this cis-acting site, a series of mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 kb subchromosome, was found to be defective in the bipolar positioning of oriC. The creation of deletion mutants allowed the identification of migS, a 25 bp sequence, as the cis-acting site for the bipolar positioning of oriC. When migS was located at the replication terminus, the chromosomal segment showed bipolar positioning. migS was able to rescue bipolar migration of plasmid DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the migS sequence on a plasmid in trans inhibited the bipolar positioning of oriC. Taken together, these findings indicate that migS plays a crucial role in the bipolar positioning of oriC. In addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and migS mutants suggests that bipolar positioning of the replicated oriC contributes to nucleoid organization.     

Conservation of sequences adjacent to the telomeric C4A2 repeats of ciliate macronuclear ribosomal RNA gene molecules.

We sequenced and compared the telomeric regions of linear rDNAs from vegetative macronuclei of several ciliates in the suborder Tetrahymenina. All telomeres consisted of tandemly repeated C4A2 sequences, including the 5' telomere of the 11 kb rDNA from developing macronuclei of Tetrahymena thermophila. Our sequence of the 11 kb 5' telomeric region shows that each one of a previously described pair of inverted repeats flanking the micronuclear rDNA (Yao et al., Mol. Cell. Biol. 5: 1260-1267, 1985) is 29 bp away from the positions to which telomeric C4A2 repeats are joined to the ends of excised 11 kb rDNA. In general we found that the macronuclear rDNA sequences adjacent to C4A2 repeats are not highly conserved. However, in the non-palindromic rDNA of Glaucoma, we identified a single copy of a conserved sequence, repeated in inverted orientation in Tetrahymena spp., which all form palindromic rDNAs. We propose that this sequence is required for a step in rDNA excision common to both Tetrahymena and Glaucoma.     

Allele-specific,selective amplification of a ribosomal RNA gene in tetrahymena thermophila

The amplification of ribosomal DNA during development of the somatic macronucleus In Tetrahymens thermophila was analyzed by genetic and molecular biological techniques. We have Identified an alternate form of the rDNA, structurally distinguishable from the wild-type by an extra cutting site for Bam HI in its nontranscribed spacer. The altered rDNA was Inherited in crosses in a simple Mendelian fashion, consistent with the presence of only one rRNA gene copy per haploid genome in the micronucleus. We therefore define a locus for the rRNA structural gene, the rdnA locus, with the allele determining the alternate form designated rdnA1. In over 95% of T. thermophila clones heterozygous for the rdnA locus in the micronucleus (rdnA1/rdn⁺), the macronucleus, which develops from a division product of this micronucleus, contained almost exclusively rdnA1-type amplified palindromic rDNA molecules. The rdnA1 allele is thus almost always dominant over the rdn⁺ allele with respect to amplification. This genetic variant of the rdnA locus was used to show that the single, free, nonpalindromic rRNA genes, which are synthesized during rDNA amplification, are derived from micronuclear gene copies from both chromosomal homologs. We therefore conclude that in these heterozygotes, selective amplification of the rdnA1 allele is not caused by the production of only one type of free, single rRNA gene during amplification.