摘要:

AbstractWe show that bacteriophage λ DNA fragments microinjected into the macronucleus of the ciliated protozoanParameciumcan replicate as unit‐length linear molecules. These linear DNA molecules are substrates for the addition ofParameciumtelomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division. © 1992 Wiley‐Liss

ISSN:0192-253X    

DOI:10.1002/dvg.1020130202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe macronucleus ofTetrahymenacontains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level.We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. InT. thermophilathe amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% inT pigmentosa.During conditions which increase the macronuclear DNA content, very little DNA is eliminate. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA.We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.The result indicates that in a given cell line at least one of the DNA molecules becoms extinct after 60 generations which we expect would cause the cell's extinction and restrict a clone's life to 60 generations. As this does not happen in nature, there must be some control of the copy numbers preventing their extinction during vegetative multiplication. Whether elimination increases or decreases the imbalance of genes remains to be investigated. © 1992 Wiley‐Liss, I

ISSN:0192-253X    

DOI:10.1002/dvg.1020130203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractInTetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000‐kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species ofTetrahymenausing histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley‐Liss,

ISSN:0192-253X    

DOI:10.1002/dvg.1020130204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractTransformation by microinjection of macronucleoplasm inParamecium caudatumwas investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene(cnrA+orcnrB+)of the donor 9–24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells. In unstable transformation, injected macronucleoplasm is distributed only to one of the daughter cells at every division until about the fifth to seventh fission after injection and then begins to assort equally to daughter cells. The cell cycle stage at injection may influence the mode of transformation. Interspecific microinjection of macronucleoplasm fromP. multimicronucleatumandP. tetraureliatoP. caudatum.resulted in the expression of foreign genes inP. caudatum.In one case, injection of macronucleoplasm ofP. tetraureliaproduced a stable transformant indicating replication of foreign macronucleoplasm inP. caudatum.This work reveals the mode of transformation by injected macronucleoplasm and shows the possibility of transformation amongParameciumspecies, which is significant in the study of the conservation of gene products and the mechanism of gene expression in different species. © 1992 Wiley‐Liss,

ISSN:0192-253X    

DOI:10.1002/dvg.1020130205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractDuring vegetative, asexual reproduction in heterozygousTetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf/ the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published Rfvalues are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of Rf. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with ∼45 assorting units, as has been asserted. © 1992 Wiley‐Liss,

ISSN:0192-253X    

DOI:10.1002/dvg.1020130206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes ofTetrahymenawas acquired by differentTetrahymenaspecies at different times during evolution. We have now approached the question of intron mobility experimentally by crossing intron+and intron−strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron inT. pigmentosais inherited as a neutral character and that intron+and intron−alleles segregate in a Mendelian fashion with no sign of intron homing. In an analysis of vegetatively growing cells containing intron+and intron−rDNA, initially in the same macronucleus, we similarly find no evidence of intron homing.During the course of this work, we observed to our surprise that progeny clones from some crosses contained three types of rDNA. One possible explanation is thatT. pigmentosahas twordnloci in contrast to the single locus found inT. thermophila.Some of the progeny clones from the genetic analysis were expanded for several hundred generations, and allelic assortment of the rDNA was demonstrated by subcloning analysis. © 1992 Wiley‐L

ISSN:0192-253X    

DOI:10.1002/dvg.1020130207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractAn in vitro transcription system was established using extrachromosomal nucleoli fromTetrahymena pyriformismacronuclei as a template. Ribosomal precursor RNA (pre‐rRNA) and nascent pre‐rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract fromTetrahymena thermophila.The transcription product was examined for the presence of transcripts fromT. pyriformisribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from aT. pyriformisrDNA clone. A sequence difference betweenT. pyriformisandT. thermophilain the 5′ region of their 35S pre‐rRNAs permitted exclusive detection ofT. pyriformistranscripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of tran‐scriptional control during development and differentiation. © 1992 Wiley

ISSN:0192-253X    

DOI:10.1002/dvg.1020130208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractDense‐core granules represent an adaptation of specialized secretory cell to facilitate stimulus‐regulated release of stored proteins. Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates. InTet‐rahymena thermophila, the ability to generate mutants in dense‐core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis. We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors. We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild‐type cells. The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes. In addition, the assay represents a powerful technique for diagnosis of new mutants. © 1992 Wiley

ISSN:0192-253X    

DOI:10.1002/dvg.1020130209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractUnder appropriate conditions, Alcian Blue‐induced exocytosis ofTetrahymenamucocysts leads to formation of a capsule that surrounds the cell. This phenomenon is an example of regulated secretion, a mechanism of fundamental significance in eukaryotic cells. In order to dissect genetically the mechanism of mucocyst biogenesis and regulated exocytosis, mutants unable to form capsules (Caps–) were isolated. In this paper we report a genetic characterization of Caps– mutants in this collection. The mutations in mutants SB255 and SB281 behave as single recessive Men‐delian mutations. The mutation in SB251 is restricted to the macronucleus, and could not be further characterized by the genetic methods we used. Complementation tests suggest the existence of at least 2 genes, namedexoAandexoB; additional mutant loci are likely to be included in the mutant collection. Deletion mapping using nulli‐somic strains showed thatexoAandexoBare located on the left arm of chromosome 4. Theexo‐3mutation, which behaves as recessive and complements withexoA1in SB255 andexoB2in SB281, maps to chromosome 3. These Caps– mutants may be useful for the elucidation of the developmental pathway of mucocyst biogenesis and the control of regulated secretion in eukaryotic cells. © 1992 W

ISSN:0192-253X    

DOI:10.1002/dvg.1020130210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS‐1, ofTetrahymena thermophilablocked in secretion of lysosomal acid hydrolases have been analyzed. Although blocked constitu‐tively in secretion, MS‐1 contains active lysosomal hydrolases in amounts equivalent to the wild type. The 3:1 segregation in F‐2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec. The sec allele of MS‐1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength. Deletion mapping by crossing MS‐1 with nullisomic strains, all secreting lysosomal hydrolases at wild‐type rates, was performed. The sec phenotype was expressed in monosomic‐4 progeny only, indicating that the sec allele is located on chromosome 4 of T. thermophila. © 1992

ISSN:0192-253X    

DOI:10.1002/dvg.1020130211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY