Does ribosomal DNA get out of the micronuclear chromosome in Paramecium tetraurelia by means of a rolling circle?

The macronuclear genes coding for rRNA (ribosomal DNA [rDNA]) of Paramecium tetraurelia, stock 51, are arranged in polymers consisting of units made up of a transcribed coding region and a nontranscribed spacer region. The whole macronuclear polymer ends with a portion of the spacer on either end followed by a telomere. Six kinds of macronuclear units, or genes, were mapped. Spacers were different, and transcribed regions were the same. These genes are found in markedly different numbers in the macronucleus. The most common gene shows two regions in the spacer where a sequence is followed by a direct repeat. The next most common gene is similar but shows a deletion plus a number of base pair substitutions. Although most cosmid clones contain only a single kind of gene, many contain more than one. These are thought to be produced by somatic crossing over. The four micronuclear genes that have been isolated consist of a single central transcribed region and portions of the spacer on either end. Sequencing indicates that the two ends of the molecule are partially redundant. While the spacer region at the right end of the macronuclear polymer is derived from the micronuclear spacer on the right, the spacer at the left end of the macronuclear polymer is derived from regions of the micronuclear spacer on both the right and the left. To account for this situation, a rolling-circle model for generation of the macronuclear rDNA from the micronuclear DNA is proposed.

Structure and expression of genes for surface proteins in Paramecium.

Surface proteins from 11 antigenic types of Paramecium tetraurelia vary in molecular weight from 251,000 to 308,000. The size of a series of polyadenylated RNAs obtained from these types were correlated with the sizes of the proteins and judged to be the mRNAs for the proteins. The mRNAs were used to identify genomic DNA clones containing complementary sequences. The gene for antigen A was present in one copy per genome, and the data suggest that extensive introns were absent. When restriction enzyme digests of DNA from cultures of paramecia with active and inactive genes were probed with portions of the cloned genes, no evidence for rearrangements or changes in gene dosage was found.

Mutants affecting processing of DNA in macronuclear development in paramecium.

In Paramecium tetraurelia, stock 51, the A surface protein is coded by the wild type A51 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 d12 mutant d12(-1300) is homozygous for the allele A-1300 and the mutant d12(+1) for A+1, analysis by the polymerase chain reaction indicates that the micronuclei 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 d12 allele A-1300 was originally determined to extend from position -1300 (relative to the start of translation of the A gene) to the end of the chromosome. Later, a derivative of this strain, homozygous for the d12 allele A+1 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 d12 allele and one homozygous for the wild-type A51 allele. Previous work on the non-Mendelian d48 mutant (which has intact A51 genes in its micronucleus, but has truncated A51 genes in its macronucleus) has shown that intact A51 alleles must be present in the old macronucleus in order for A51 alleles to undergo proper processing. We find that d12 alleles act on A51 alleles in heterozygotes such that intact macronuclear A genes are no longer required for proper processing of A51. Thus, in crosses of 51 x d12 (either +1 or -1300) d12 exconjugants, as well as 51 exconjugants, give rise to clones carrying both intact A51 and truncated d12 alleles. Remarkably the d12 alleles, which are themselves deleted during processing, are capable in the heterozygote of fostering normal processing of the A51 allele.

mRNAs for the immobilization antigens of Paramecium.

Immobilization antigens of stock 51 of Paramecium tetraurelia were subjected to electrophoresis in NaDodSO(4)/polyacrylamide gels. Type A is estimated to have a molecular size of 300,000 daltons; H is estimated to be 288,000, D to be 280,000, E to be 270,000, B to be 253,000, and C to be 250,000. Poly(A)(+)RNAs have been isolated from cells producing these antigens and subjected to electrophoresis in methylmercury gels. A major band is found to vary in mobility with antigenic type: Its position in preparations derived from paramecia synthesizing antigen A indicates a size of 8400 nucleotide residues; its position from paramecia synthesizing other antigens indicate H, 8200; D, 7900; E, 7500; B, 7600; and C, 7000. Because of the sizes and quantities of these RNAs, it is argued that they probably represent the mRNAs for the immobilization antigens. It is concluded that each immobilization antigen probably consists of a single polypeptide and that only one major serotype-determining mRNA is present in each antigenically different paramecium.

Micronuclear DNA from Paramecium tetraurelia: serotype 51 A gene has internally eliminated sequences.

A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all of the unbroken cells and macronuclei and approximately two thirds of the macronuclear fragments. Next a higher speed centrifugation of 9,000 g sediments the micronuclei and frees them from small particulates and soluble constituents. Advantage is then taken of the fact that micronuclei have a lower density than do macronuclear fragments in 45%-60% Percoll. Micronuclei float to the top during centrifugation at 24,000 g, while macronuclear fragments sediment. After several cycles of centrifugation in Percoll, the micronuclei, although heavily contaminated with cytoplasmic components, are essentially free of macronuclei and macronuclear fragments. Micronuclear DNA can then be extracted from the suspension. The whole procedure is very rapid and in about an hour micronuclear and macronuclear DNA can be separated. About 2 micrograms of micronuclear DNA can be obtained from 6 x 10(7) paramecia. We find that there are internal sequences in the micronuclear A gene DNA in wild type cells which are eliminated when the micronuclei develop into macronuclei. They yield unique restriction fragments for micronuclei and macronuclei. Therefore the purity of the preparations is easily monitored by probing Southern blots of restriction enzyme-digested DNA with the cloned A gene. No differences have been found between the micronuclear A gene in wild type and the d48 mutant.

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.

Developmentally excised sequences in micronuclear DNA of Paramecium.

DNA processing occurs in ciliates at autogamy and conjugation when new macronuclei are formed from micronuclei and old macronuclei degrade. Processing of micronuclear DNA consists of removal of certain internal sequences, chromosomal fragmentation, addition of new telomeres, and amplification. Aside from a recent brief report, internal eliminated sequences have not been described in Paramecium. In this paper we characterize nine internal eliminated sequences found within and near the gene that codes for surface protein A in Paramecium tetraurelia. Of these nine, seven are located within the translated portion of the gene, and all include short, inverted terminal repeats. The characteristic sequence, TA, appears at the boundaries of all of the internal eliminated sequences.

Deviation from the universal code shown by the gene for surface protein 51A in Paramecium.

The immobilization antigens (i-antigens) of Paramecium, large polypeptides of relative molecular mass approximately 300,000, are located on the cell surface. Each i-antigen is encoded by a different unlinked gene, and no more than one gene is expressed at a time. The proteins and the mRNAs and genes encoding them are readily isolated. Here we report the nucleotide sequence of three regions of the A i-antigen gene from stock 51 of Paramecium tetraurelia. Surprisingly, all reading frames contain TAA and TAG stop codons, even though there is evidence that one reading frame of these sequences codes for the i-antigen. We suggest that in Paramecium UAA and UAG code for amino acids, instead of serving as translational stops as they do in all other organisms.

Transformation of Paramecium by microinjection of a cloned serotype gene.

Paramecia of a given serotype express only one of several possible surface proteins called immobilization antigens (i-antigens). A 16-kilobase plasmid containing the gene for immobilization antigen A from Paramecium tetraurelia, stock 51, was injected into the macronucleus of deletion mutant d12, which lacks that gene. Approximately 40% of the injected cells acquired the ability to express serotype A at 34 degrees C. Expression appeared to be regulated normally. The transformed cells, like wild type, could be switched to serotype B by antiserum treatment and culture at 19 degrees C; on transfer to 34 degrees C, they switched back to serotype A expression. Many of the lines retained the ability to express serotype A until autogamy, when the old macronucleus is replaced by a new one derived from the micronucleus. DNA from transformants contained the injected plasmid sequences, which were replicated within the paramecia. No evidence for integration was obtained. The majority of replicated plasmid DNA comigrated with a linearized form of the input plasmid. Nonetheless, the pattern of restriction fragments generated by transformant DNA and that generated by input plasmid DNA are identical and consistent with a circular rather than a linear map. These conflicting observations can be reconciled by assuming that a mixture of different linear fragments is present in the transformants, each derived from the circular plasmid by breakage at a different point. Copy-number determinations suggest the presence of 45,000-135,000 copies of the injected plasmid per transformed cell. These results suggest that the injected DNA contains information sufficient for both controlled expression and autonomous replication in Paramecium.