The DNA of ciliated protozoa.

DNA was isolated from macronuclei and micronuclei of the ciliated protozoan, Stylonychia mytilus under conditions that minimize the possibility of DNA degradation Macronuclear DNA has an S value of 10 to 11 in sucrose gradients Macronuclear DNA has an average molecular weight of 115×106 daltons and a range of molecular weights of 10×106 to 195×106 daltons The average length of macronuclear DNA, measured by electron microscopy, is 080 microns and the range is 02 to 22 microns Almost all micronuclear DNA pieces are too long to be measured by electron microscopy The shortest piece of micronuclear DNA found was 150 microns in length

DNA of ciliated protozoa

Publisher Summary This chapter discusses methods in Paramecium research. Paramecium and other eukaryotic unicells occupy a strategically important level of biological structure and function between the levels of prokaryotic and multicellular organisms. Paramecium possesses many attractive research features, some of them are (1) it is readily obtainable from nature, from commercial sources, and from investigators. (2) It is readily cultivated in isolation by methods varying from the simplest and crudest to fully defined axenic culture, and several others. Methods used in research on the general biology and genetics of P. aurelia were set forth by Sonneborn. This chapter updates the summary and extends it to all species of Paramecium. Paramecium caudatum, P. multimicronucleatum, P. aurelia, and P. bursaria occur commonly, and P. trichium and P. polycaryum less commonly, in still and moving fresh-water. For ecological and other studies that require sampling of natural sources Hairston and Kellerman exploited the long-known fact that paramecia swim toward the cathode in a weak electric current. The results of genetic work on higher organisms led to the concept of the gene that made possible the successful attack on the molecular genetics of bacteria and viruses.

Chapter 12 Methods in Paramecium Research

The immobilization antigens (i-antigens) of Paramecium, large polypeptides of relative molecular mass ∼300,000 (ref. 1), 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 isolated2,3. 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.

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

The germline genomes of ciliated protozoa are dynamic structures, undergoing massive DNA rearrangement during the formation of a functional macronucleus. Macronuclear development involves chromosome fragmentation coupled with de novo telomere synthesis, numerous DNA splicing events that remove internal segments of DNA, and, in some ciliates, the reordering of scrambled gene segments. Despite the fact that all ciliates share similar forms of DNA rearrangement, there appears to be great diversity in both the nature of the rearranged DNA and the molecular mechanisms involved. Epigenetic effects on rearrangement have also been observed, and recent work suggests that chromatin differentiation plays a role in specifying DNA segments either for rearrangement or for elimination.

Genome Remodeling in Ciliated Protozoa

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.

Structure and expression of genes for surface proteins in Paramecium.

https://www.genetics.org/content/genetics/33/4/349.full.pdf

A Study of Some Properties of the Cytoplasmic Factor, "Kappa," in Paramecium Aurelia, Variety 2.

The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its surface. Although the A gene is intact in the micronuclei of d48, the A gene copies in the macronucleus contain a large deletion eliminating virtually the entire coding sequence. Previous studies showed that microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently restored A expression after autogamy. Together with other data, this result suggests that in wild type cells the A gene in the old macronucleus ensures the presence of a cytoplasmic factor that prevents A gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular mechanisms involved in this unusual phenomenon, we attempted to define the region(s) of the A gene necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments of the A gene have the ability to rescue d48 to different extents, some being more efficient than others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might act as or encode a cytoplasmic factor. Molecular mechanisms that may be involved in the rescue of d48 are discussed.

https://www.genetics.org/content/genetics/129/3/727.full.pdf

Permanent rescue of a non-Mendelian mutation of Paramecium by microinjection of specific DNA sequences.

The non-Mendelian mutant d48 of Paramecium tetraurelia contains micronuclear wild type A genes, but at autogamy and conjugation proper processing fails and new macronuclei lack A genes. When cloned A genes are injected into the macronucleus of d48, proper processing is restored at the next autogamy; d48 is rescued, becoming permanently wild type. In the present study we have injected portions of the A gene into d48. We find that the ability to rescue extends over a large portion of the gene, with highest activity near a series of 221-bp repeat units in the middle of the gene. Regions outside the A gene are inactive.

https://www.genetics.org/content/genetics/136/4/1325.full.pdf

Identification of DNA segments capable of rescuing a non-mendelian mutant in paramecium.

A family of genes is responsible for production of surface antigenic components of Paramecium tetraurelia. These surface proteins are expressed in a mutually exclusive manner. Individuals rarely display more than one type. However, changes in environmental conditions can cause different surface proteins which replace preexisting types to be expressed. We investigated the nature of regulation of the genes for the A, C, and H surface antigens of P. tetraurelia. A system for in vitro run-on transcription was developed from crude Paramecium extracts and used in this analysis. The genes for surface antigens A and H were controlled at the level of transcription. However, the gene for surface antigen C demonstrated both transcriptional and posttranscriptional control, depending on the serotype being expressed. When animals expressed serotype A, the gene for surface antigen C was not transcribed. However, when animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable surface antigen C mRNA was present in the cells, although surface antigen C was not detectable by serotype testing or by a salt-alcohol extraction method. The kinetics of transformation from serotype H to serotype C were determined by using the in vitro transcription system and monitoring steady-state RNA levels. During the transition, serotype A transcription was detected in run-on transcription experiments, although this RNA did not accumulate. The results indicate that serotype expression is controlled at several levels and that not all serotype genes are controlled in the same manner.

J R Preer

Papers

Structure and expression of genes for surface proteins in Paramecium.

A Study of Some Properties of the Cytoplasmic Factor, "Kappa," in Paramecium Aurelia, Variety 2.

Permanent rescue of a non-Mendelian mutation of Paramecium by microinjection of specific DNA sequences.

Identification of DNA segments capable of rescuing a non-mendelian mutant in paramecium.

Multilevel regulation of surface antigen gene expression in Paramecium tetraurelia.