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1.
Wickert S  Nangle L  Shevel S  Orias E 《Genetics》2000,154(3):1155-1167
The genetics of the ciliate Tetrahymena thermophila are richer than for most other eukaryotic cells, because Tetrahymena possesses two genomes: a germline (micronuclear) genome that follows a Mendelian model of genetic transmission and a somatic (macronuclear) genome, derived from the micronuclear genome by fragmentation, which follows a different genetic transmission model called phenotypic assortment. While genetic markers in the micronucleus fall into classical linkage groups under meiotic recombination and segregation, the same markers in the macronucleus fall into coassortment groups (CAGs) under phenotypic assortment by the random distribution of MAC chromosome pieces. We set out to determine whether genomic mapping in the macronucleus by genetic means is feasible. To investigate the relationship between the micronuclear map and coassortment groups, we systematically placed into CAGs all of the markers lying on chromosome 1L that are also found in the macronucleus. Sixteen CAGs were identified, 7 of which contain at least two loci. We have concluded that CAGs represent a fundamental genetic feature of the MAC. The MIC and MAC maps on 1L are colinear; that is, CAGs consist exclusively of markers that map to a continuous segment in a given region of the micronuclear map, with no intervening markers from other CAGs. These findings provide a solid foundation for exploiting the MAC chromosome pieces to build a physical map of the Tetrahymena genome.  相似文献   

2.
The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific, directed by a conserved chromosome breakage sequence (Cbs element). An accompanying article in this issue reports the development of a successful scheme for the genome-wide cloning and identification of functional chromosome breakage sites. This article reports the physical and genetic characterization of 30 functional chromosome breakage junctions. Unique sequence tags and physical sizes were obtained for the pair of macronuclear chromosomes generated by fragmentation at each Cbs. Cbs-associated polymorphisms were used to genetically map 11 junctions to micronuclear linkage groups and macronuclear coassortment groups. Two pairs of junctions showed statistically significant similarity of the sequences flanking the Cbs, suggestive of relatively recent duplications of entire Cbs junctions during Tetrahymena genome evolution. Two macronuclear chromosomes that lose at least one end in an age-related manner were also identified. The whole-genome shotgun sequencing of the Tetrahymena macronucleus has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from natural ends of macronuclear chromosomes, Cbs junctions will provide useful sequence tags for relating macro- and micronuclear genetic, physical, and whole-genome sequence maps.  相似文献   

3.
4.
The focus of this review is on the micronucleus and macronucleus in the ciliated protozoa and the organization and function of the DNA molecules within them. We present (1) some of the structural and functional differences which are known, (2) the genetic evidence for macronuclear units, (3) two hypotheses for the organization of the DNA molecules in the macronucleus to explain these units, and (4) experiments designed to discriminate between these hypotheses. We conclude that the size of the genome is not reduced in the macronucleus and that there are 45 copies of the haploid genome present in the macronucleus of normal strains of Tetrahymena pyriformis and 800 copies in the macronucleus of Paramecium aurelia. The ciliate genome is relatively simple in terms of repeated sequences. However, not all copies of the genes present in the macronucleus may be identical since fractions of differing thermal stability appear after renaturation.The work reported in this paper has been supported by a research grant, GM 15879, from the National Institute of General Medical Sciences, U.S. Public Health Service, and by grants from the Science and Medical Research Councils of Great Britain. We are also grateful to the Center for Human Growth and Development, University of Michigan, for a summer fellowship facilitating collaboration.  相似文献   

5.
In Tetrahymena, 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 of Tetrahymena using 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.  相似文献   

6.
We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila. To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events. This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome. Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs. We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development. The variable somatic genomes so produced may be the source of phenotypically variant cell lines.  相似文献   

7.
Programmed chromosome breakage occurs at 50–200 specific sites in the genome of Tetrahymena thermophila during somatic nuclear (macronuclear) differentiation. Previous studies have identified a 15 bp sequence, the Cbs (for chromosome breakage sequence), that is necessary and sufficient to specify these sites. In this study we determined the effects of mutations in the Cbs on its ability to specify the chromosome breakage site and promote new telomere formation in conjugating cells. Twenty-one constructs with single nucleotide substitutions covering all 15 positions of the Cbs were made and tested. Fourteen of them (covering 11 positions) abolished breakage entirely, six (covering six positions, including the remaining four) caused partial loss of breakage function and one showed no detectable effect. This result indicates that the Cbs has an exceptionally long and stringent sequence requirement. It offers no evidence that the Cbs contains a separate domain for promoting new telomere formation. In addition, we found that a partially functional Cbs retained in the macronucleus does not induce chromosome breakage during vegetative growth and that excess copies of this germline-specific sequence in the somatic nucleus have little deleterious effect on cell growth.  相似文献   

8.
Synopsis.
A satisfactory model of the Tetrahymena thermophila macronucleus must explain its genetic behavior in terms of its constituent molecules. Particular genetic phenomena requiring explanation are (a) phenotypic assortment , here interpreted as resulting from allelic disjunction rather than from differential gene expression; (b) unequal allelic input for some loci , interpreted as a consequence of unequal and selective replication of some alleles during early macronuclear development; (c) delayed assortment at some loci , interpreted as an effect of inequality of allelic input combined with a generalized elevation of DNA content during early clonal history; (d) linkage disruption , probably reflecting continuous somatic recombination rather than dissolution of chromosomes into small repliconic units; (e) assortment depression , brought about by the occasional association of homologous replicons (chromosomes) or else by a differential increase in some classes of replicons; (f) ploidy-related developmental differences in macronuclear primordia are interpreted on the basis of quantitative differences in DNA rather than in terms of an early perception of genic imbalance, (g) Ploidy independent macronuclear DNA content is consistent with several models of size regulation.  相似文献   

9.
Tetrahymena micronuclear DNA fragments have been cloned in the plasmid pBR322. One clone, pTt 2512, has been found to contain the C-C-C-C-A-A hexanucleotide repeat which is also present in the macronuclear rDNA. Further restriction enzyme digestion and hybridization studies suggest that the clone also contains sequences that are not present in the somatic macronucleus. The flanking sequences of the C4A2 repeats in this clone were separated into four restriction fragments, one from one side and three from the other. These fragments were used as probes for Southern hybridization to study the organizations of similar sequences in the macronucleus and micronucleus. All four fragments hybridized to many fragments of restriction enzyme digested micronuclear DNA. However, none of these hybridizations were detected in the macronucleus. Thus, these families of repetitive DNA are completely eliminated from the macronucleus. Further analysis suggested that the four different sequences may be linked at other locations of the genome. Using nullisomic strains of Tetrahymena, it is found that at least one of these sequences is present in more than one chromosome. Studies of various normal and star strains of Tetrahymena suggest that these sequences are stable in the normal micronucleus but are altered drastically in the defective micronuclei of the star strains. Eliminated DNA of similar nature has also been found in at least five other randomly selected clones of micronuclear DNA and may be present widely in the genome.  相似文献   

10.
Ciliates are microbial eukaryotes that separate their nuclear functions into a germline micronucleus and a somatic macronucleus. During development of the macronucleus the genome undergoes a series of reorganization events that includes the precise excision of intervening DNA. Here, we determine the architecture of four loci in the micronuclear and macronuclear genomes of the ciliate Chilodonella uncinata and compare the levels of variation in micronuclear-limited sequences to macronuclear destined sequences at two of these loci. We find that within a population, germline-limited sequences are evolving at the same rate as other putatively neutral sites, but between populations germline-limited sequences are accumulating mutations at a much faster rate than other sites. We also find evidence of macronuclear recombination and incomplete elimination of intervening DNA, which result in increased diversity in the macronuclear genome. Our results support the assertion that the unusual genomic features of ciliates can result in rapid and unpredicted patterns of diversification.  相似文献   

11.
Extreme stress situations can induce genetic variations including genome reorganization. In ciliates like Tetrahymena thermophila, the approximately 45‐fold ploidy of the somatic macronucleus may enable adaptive responses that depend on genome plasticity. To identify potential genome‐level adaptations related to metal toxicity, we isolated three Tetrahymena thermophila strains after an extended adaptation period to extreme metal concentrations (Cd2+, Cu2+ or Pb2+). In the Cd‐adapted strain, we found a approximately five‐fold copy number increase of three genes located in the same macronuclear chromosome, including two metallothionein genes, MTT1 and MTT3. The apparent amplification of this macronuclear chromosome was reversible and reproducible, depending on the presence of environmental metal. We also analysed three knockout (KO) and/or knockdown (KD) strains for MTT1 and/or MTT5. In the MTT5KD strain, we found at least two new genes arising from paralogous expansion of MTT1, which encode truncated variants of MTT1. The expansion can be explained by a model based on somatic recombination between MTT1 genes on pairs of macronuclear chromosomes. At least two of the new paralogs are transcribed and upregulated in response to Cd2+. Altogether, we have thus identified two distinct mechanisms, both involving genomic plasticity in the polyploid macronucleus that may represent adaptive responses to metal‐related stress.  相似文献   

12.
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.  相似文献   

13.
14.
The DNA of ciliated protozoa.   总被引:35,自引:0,他引:35       下载免费PDF全文
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.  相似文献   

15.
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.  相似文献   

16.
The ciliated protozoa Tetrahymena contains two nuclei, a micronucleus and a macronucleus. In the vegetatively growing cell, the macronucleus divides amitotic while the micronucleus divides by mitosis. It has been indicated that microtubules are involved in macronuclear division and microtubules are observed to exist in the dividing macronucleus. To clarify the localization and the organization of microtubules in the amitotic dividing macronuclei, we used immunofluorescent staining technique. The microtubules were observed in the cytoplasm and macronucleus. The microtubules were organized and dynamically changed their distribution throughout the macronuclear division. We suggest a possibility that these microtubules are involved in 'amitotic' distribution of chromatin throughout the macronuclear division.  相似文献   

17.
Endo M  Sugai T 《Zoological science》2011,28(7):482-490
The macronucleus of the ciliate Tetrahymena cell contains euchromatin and numerous heterochromatins called chromatin bodies. During cell division, a chromatin aggregate larger than chromatin body appears in the macronucleus. We observed chromatin aggregates in the dividing macronucleus in a living T. thermophila cell, and found that these were globular in morphology and homogeneous in size. To observe globular chromatin clearly, optimal conditions for making it compact were studied. Addition of Mg ion, benomyl and oryzalin, microtubule inhibitors, to cell suspension was effective. Globular chromatin appeared when the micronuclear anaphase began at the cell cortex, and disappeared long after cell separation. Using living cells with a small macronucleus at early log phase, we counted the number of globular chromatin per nucleus and measured the DNA content of globular chromatin in the macronucleus which was stained with Hoechst 33342 by using ImageJ. The number of globular chromatin per nucleus was reduced by half after division, indicating the globular chromatin is a distribution unit of DNA. A globular chromatin contained similar DNA content as that of the macronuclear genome. We developed methods for inducing and isolating a cell with an extremely small macronucleus with a DNA amount of one globular chromatin. These cells grew, divided, and give clones, suggesting that the macronuclear genome is not dispersed within the macronucleus and the globular chromatin may be a macronuclear genome. We named this globular chromatin "macronuclear genome unit" (MGU).  相似文献   

18.
R. S. Coyne  M. C. Yao 《Genetics》1996,144(4):1479-1487
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.  相似文献   

19.
The objective of this study was to test the idea that the macronuclear (somatic) genetic information is randomly distributed at each cell division in Tetrahymena pyriformis, syngen 1. We took advantage of a quick and reliable test for the detection of stable vegetative segregants in clones heterozygous for ts2, a heat-sensitive mutation. Clones that originated from cells inferred to contain very few (1-3) copies of the ts2+ allele in their macronuclei were selected for pedigree analysis. Experimental results were compared with the results of a computer simulation of the experiment. Our results are fully consistent with the predictions of the Allen-Nanney-Schensted model concerning the replication and distribution of functional gene copies. This model proposes that the macronucleus contains many functional copies of a given gene that are duplicated once during the cell cycle and are randomly distributed to the two-daughter nuclei. Our work has provided a more sensitive test of the assumption of randomness than was previously available. Our evidence for complete randomness suggests that 45, the effective number of macronuclear gene copies previously inferred from the segregation rate, indeed represents the actual number of copies. This conclusion, coupled with previously available evidence that the macronucleus is approximately 45-ploid, suggests that the segregating genetic units in the macronucleus are in effect haploid. This appears to remove the need to postulate inter-allelic repression to account for the phenomenon of phenotypic assortment. Our results, as well as those of others, also are inconsistent with any simple form of the master-slave hypothesis of Allen and Gibson.  相似文献   

20.
潘惟钧  陶英 《遗传学报》1991,18(2):127-131
形态学和遗传学方法早巳证明四膜虫接合过程中旧大核退化消失,其基因型对接合后代不发生影响。本文以1种具有强大复制优势而且是抗药性的rDNA分子,rdna-A3,为指标,证明在接合过程中旧大核内上万个rDNA分子没有1个能进入新大核,从而在基因分子水平上证明旧大核的退化是极为彻底的。同时检测了接合过程中旧大核内DNA发生降解的时间。  相似文献   

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