Variable copy number of macronuclear DNA molecules in Tetrahymena

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. © 1992 Wiley-Liss, Inc.     

Variable copy number of macronuclear DNA molecules in Tetrahymena.

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.     

Division and Formation of the Macronuclei of Keronopsis rubra*

The hypotrichous ciliate Keronopsis rubra has ～10 micronuclei and ～100 small macronuclei. DNA synthesis proceeds synchronously in all macronuclei in the 2nd half of the cell cycle which takes about 24 hr at room temperature. A G₂ phase is virtually absent, each nucleus dividing as soon as the replication band has passed over it. The micronuclear S phase falls within macronuclear G₁ and is followed by immediate division. Comparative cytophotometric measurements of Feulgen-stained preparations indicate that the DNA content of G₁ macronuclei is scattered widely in a skewed normal distribution, with a peak corresponding to the DNA content of a G₁ micronucleus. Measurements of dividing macronuclei indicate unequal distribution of DNA between daughter nuclei and lead to the conclusion that the units of assortment must be smaller than whole genomes unless the micronucleus is polyploid. After conjugation, a large macronuclear anlage with threads resembling split prophase chromosomes is formed. The threads condense and pass singly into the cytoplasm where they are thought to give rise to the numerous small macronuclei of the vegetative cells.     

A Chimeric Chromosome in the Ciliate Oxytricha Resulting from Duplication

In a process similar to exon splicing, ciliates use DNA splicing to produce a new somatic macronuclear genome from their germline micronuclear genome after sexual reproduction. This extra layer of DNA rearrangement permits novel mechanisms to create genetic complexity during both evolution and development. Here we describe a chimeric macronuclear chromosome in Oxytricha trifallax constructed from two smaller macronuclear chromosomes. To determine how the chimera was generated, we cloned and sequenced the corresponding germline loci. The chimera derives from a novel locus in the micronucleus that arose by partial duplication of the loci for the two smaller chromosomes. This suggests that an exon shuffling-like process, which we call MDS shuffling, enables ciliates to generate novel genetic material and gene products using different combinations of genomic DNA segments.     

Autonomously replicating macronuclear DNA pieces are the physical basis of genetic coassortment groups in Tetrahymena thermophila

The macronucleus of the ciliate Tetrahymena thermophila contains a fragmented somatic genome consisting of several hundred identifiable chromosome pieces. These pieces are generated by site-specific fragmentation of the germline chromosomes and most of them are represented at an average of 45 copies per macronucleus. In the course of successive divisions of an initially heterozygous macronucleus, the random distribution of alleles of loci carried on these copies eventually generates macronuclei that are pure for one allele or the other. This phenomenon is called phenotypic assortment. We have previously reported the existence of loci that assort together (coassort) and hypothesized that these loci reside on the same macronuclear piece. The work reported here provides new, rigorous genetic support for the hypothesis that macronuclear autonomously replicating chromosome pieces are the physical basis of coassortment groups. Thus, coassortment allows the mapping of the somatic genome by purely genetic means. The data also strongly suggest that the random distribution of alleles in the Tetrahymena macronucleus is due to the random distribution of the MAC chromosome pieces that carry them.     

Mitosis-like macronuclear division in a ciliate

The macronucleus of a small marine ciliate of the genus Protocrucia consists of a cluster of ten vesicles which give rise to 20 distinct chromosomal elements in the course of prophase-like condensation stages. Size differences of vesicles and chromosomes are cytological indications of their genetic individuality. In an anaphase-like stage, the chromosomal elements are separated in two daughter groups which re-form 10 vesicles each. The micronucleus divides simultaneously. The existence of a precisely functioning mode of chromosome distribution is also indicated by DNA measurements. Since the macronucleus contains much more DNA than the micronucleus, the macronuclear chromosomes are thought to be oligotenic. This hypothesis is supported by the much larger size of the macronuclear chromosomes. In contrast to other modes of macronuclear division known so far, this ciliate has retained some essential features of mitosis.     

Polymorphism, recombination and alternative unscrambling in the DNA polymerase alpha gene of the ciliate Stylonychia lemnae (Alveolata; class Spirotrichea)

DNA polymerase alpha is the most highly scrambled gene known in stichotrichous ciliates. In its hereditary micronuclear form, it is broken into >40 pieces on two loci at least 3 kb apart. Scrambled genes must be reassembled through developmental DNA rearrangements to yield functioning macronuclear genes, but the mechanism and accuracy of this process are unknown. We describe the first analysis of DNA polymorphism in the macronuclear version of any scrambled gene. Six functional haplotypes obtained from five Eurasian strains of Stylonychia lemnae were highly polymorphic compared to Drosophila genes. Another incompletely unscrambled haplotype was interrupted by frameshift and nonsense mutations but contained more silent mutations than expected by allelic inactivation. In our sample, nucleotide diversity and recombination signals were unexpectedly high within a region encompassing the boundary of the two micronuclear loci. From this and other evidence we infer that both members of a long repeat at the ends of the loci provide alternative substrates for unscrambling in this region. Incongruent genealogies and recombination patterns were also consistent with separation of the two loci by a large genetic distance. Our results suggest that ciliate developmental DNA rearrangements may be more probabilistic and error prone than previously appreciated and constitute a potential source of macronuclear variation. From this perspective we introduce the nonsense-suppression hypothesis for the evolution of ciliate altered genetic codes. We also introduce methods and software to calculate the likelihood of hemizygosity in ciliate haplotype samples and to correct for multiple comparisons in sliding-window analyses of Tajima's D.     

Evidence for Chromosomal Macronuclear Substructures in Tetrahymena*†

SYNOPSIS.
Under the growth conditions employed, the G₁ macronucleus of Tetrahymena pyriformis HSM contains 7.4 × 10^-12 g DNA, the G₂ micronucleus 0.42 × 10^-12 g. DNA content from the Tetrahymena thermophila macronucleus did not significantly differ from that of HSM, but the micronucleus contained about twice as much DNA as the micronucleus of the HSM cells. The T. thermophila macronucleus contained on average enough DNA for ˜ 35 haploid micronuclear copies. A new spreading technic allowed separation of macronuclear substructures from cells of late G₂ to early G₁. Photometric determination of DNA content of 345 individual structures suggested the existence of 5 different-sized macronuclear structures with a DNA content corresponding to 2, 4, 8, and 16 × the basic values. Comparison of the DNA content of these structures with (a) mitotic micronuclear chromosomes and (b) meiotic micronuclear chromosomes of T. thermophila cells suggests that the 5 basic values of macronuclear structures derive from structures of micronuclear chromosomes. The micronuclear chromosomes of T. pyriformis may be oligotenic. It is suggested that these results further our understanding of macronuclear organization.     

Studies of mammalian chromosome replication

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G₁/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these late intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 m in all S phase cells except those at G₁/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units or chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.     

Regulation of Macronuclear DNA Content in Tetrahymena thermophila*†

Synopsis.
Unequal macronuclear division in Tetrahymena thermophila introduces variance into G1 macronuclei; unless eliminated such variance would result in continuous variation in DNA content. Analysis of G1 and G2 macronuclear variances reveals that the added variance is eliminated by action on the extremes of macronuclear DNA content. In this model (Model II), macronuclei with small amounts of DNA have an additional complete S phase, while those with large amounts of DNA skip S. From available data, chromatin extrusion is shown not to contribute significantly, if at all, to the elimination of variance. Computer simulations utilizing haploid subunits indicate that model II predictions apply reasonably well to experimental data in terms of coefficients of variation, mean DNA content, and frequency of additional and skipped S phases. The simulations reveal also that within certain constraints, particularly the thresholds for additional and skipped S phases, macronuclear assortment is unaffected by Model II regulation. The relationships between Model II and other aspects of the cell cycle are briefly discussed.     

Genetics of the Tubulin Gene Families of Physarum

The organization of the alpha- and beta-tubulin gene families in Physarum was investigated by Mendelian analysis. Restriction endonuclease-generated DNA fragments homologous to alpha- and beta-tubulin show length polymorphisms that can be used as markers for genetic mapping. Analysis of meiotic assortment among progeny of heterozygotes allowed alpha- and beta-tubulin sequence loci to be defined. There are four unlinked alpha-tubulin sequence loci (altA, altB, altC and altD) and at least three unlinked beta-tubulin sequence loci (betA, betB and betC). The alpha-tubulin loci are not linked to the beta-tubulin loci. --Segregation of tubulin sequence loci with respect to ben mutations that confer resistance to antitubulin benzimidazole drugs was used to investigate whether any members of the alpha- or beta-tubulin gene families are allelic to ben loci. The beta-tubulin sequence locus betB is allelic to the resistance locus benD, the betA locus is probably allelic to benA and the alpha-tubulin sequence locus altC may be allelic to benC. The molecular implications of benzimidazole resistance phenotypes when only one of the expressed beta-tubulin gene family members mutates to drug resistance are discussed in relation to tubulin function.     

Microsatellite variation among divergent populations of stalk-eyed flies, genus Cyrtodiopsis

Microsatellite primers are often developed in one species and used to assess neutral variability in related species. Such analyses may be confounded by ascertainment bias (i.e. a decline in amplification success and allelic variability with increasing genetic distance from the source of the microsatellites). In addition, other factors, such as the size of the microsatellite, whether it consists of perfect or interrupted tandem repeats, and whether it is autosomal or X-linked, can affect variation. To test the relative importance of these factors on microsatellite variation, we examine patterns of amplification and allelic diversity in 52 microsatellite loci amplified from five individuals in each of six populations of Cyrtodiopsis stalk-eyed flies that range from 2.2 % to 11.2% mitochondrial DNA sequence divergence from the population used for microsatellite development. We find that amplification success and most measures of allelic diversity declined with genetic distance from the source population, in some cases an order of magnitude faster than in birds or mammals. The median and range of the repeat array length did not decline with genetic distance. In addition, for loci on the X chromosome, we find evidence of lower observed heterozygosity compared with loci on autosomes. The differences in variability between X-linked and autosomal loci are not adequately explained by differences in effective population sizes of the chromosomes. We suggest, instead, that periodic selection events associated with X-chromosome meiotic drive, which is present in many of these populations, reduces X-linked variation.     

Microsatellite variation and differentiation in North Atlantic eels

We screened 11 populations of American, European, and Icelandic eels (Anguillidae) for allelic variation and genetic divergence at six polymorphic microsatellite loci. Within either of the two recognized Anguilla species in the North Atlantic (rostrata in the Americas, anguilla in Europe), population genetic structure was statistically significant but weak; fully 95% of the total genetic variation was present within geographic locales rather than distributed among them. The two Anguilla species also overlap greatly in allelic frequencies, so the available data proved ineffective for addressing hypotheses about the possible hybrid origins of some Icelandic eels. The overlapping microsatellite profiles contrast with nearly diagnostic species differences documented previously in allozymes and mtDNA. This and similar empirical findings in several other species support theoretical concerns that homoplasy (convergent evolution) in allelic states can compromise the utility of rapidly mutating microsatellite loci for certain types of microevolutionary questions regarding gene flow and species differences.     

Unequal distribution of DNA in the macronuclear division of the ciliate Euplotes eurystomus

During asexual fission in the ciliate Euplotes eurystomus, the macronucleus divides amitotically. The macronucleus was found to divide unequally, yielding sister pairs having a mean difference in DNA content of 11.6%. DNA content was determined by the Feulgen reaction using a fluorescent Schiffs reagent, and measuring fluorescence by cytophotometry. Variability in macronuclear DNA content was also examined in randomly-paired non-sister cells, and found to be greater than in sister cells. This greater variability could be due to accumulation of differences over a number of divisions, or to interclonal differences in equality of division. Two categories of non-sister cells were examined: recently divided, and parents constructed by averaging the DNA contents of progeny. Both showed similar variability in quantity of macronuclear DNA. The fact that cells surviving to divide showed no less variability in amount of DNA than cells immediately after division suggests that extremes in amounts of DNA resulting from unequal division are not selected against.     

Rare internal C4A4 repeats in the micronuclear genome of Oxytricha fallax.

Approximately 20,000 different short, linear, macronuclear DNA molecules are derived from micronuclear sequences of Oxytricha fallax after conjugation. These macronuclear DNAs are terminated at both ends by 20 base pairs of the sequence 5'-dC4A4-3'. Sequences homologous to this repeat (C4A4+) are also abundant in the micronuclear chromosomes, but most reside at their telomeres. Here we show that nontelomeric C4A4 clusters of 20 base pairs or longer exist in only a few hundred copies per micronuclear genome. This demonstrates that nearly none of the 20,000 sequence blocks of micronuclear DNA destined to be macronuclear DNA molecules can be flanked by full-length (20-base pair) C4A4 clusters, and therefore C4A4 repeats must be added to most, if not all, macronuclear telomeres during macronuclear development. Six internal micronuclear C4A4+ loci were cloned, and their structural relationships with macronuclear and micronuclear sequences were examined. The possible origins and functions of these rare, micronuclear internal C4A4 loci are discussed.     

Unequal crossingover between homologous chromosomes is not the major mechanism involved in the generation of new alleles at VNTR loci

To investigate the hypothesis that unequal exchange between homologous chromosomes is involved when new alleles are generated at VNTR loci, we used genetic linkage maps to identify flanking markers surrounding a VNTR marker locus. The minisatellite probe lambda MS1 was selected, as the hypervariable locus it detects undergoes spontaneous generation of new alleles in the germline at a rate of approximately 5%. Multipoint linkage analysis placed lambda MS1 within a cluster of polymorphic marker loci on chromosome 1p. Using the two closest flanking markers, CMM8 and YNZ2, we were able to characterize 12 new-allele events in terms of crossingover between the flanking markers. Statistical analysis of these data has allowed us to reject the model that assumes that events generating new alleles always involve unequal exchange between homologous chromosomes at meiosis.     

Linkage disequilibrium for DNA haplotypes near the cystic fibrosis locus in two South European populations

Summary Three polymorphic DNA marker loci (INT1L1, D7S23 and D7S399) map to a chromosomal region that is very close to the cystic fibrosis (CF) locus in terms of genetic distance. These marker loci have been used to analyse the linkage disequilibrium in 137CF families from two South European countries (Italy and Spain). The markers can be analysed for differences in linkage disequilibrium more easily in these populations than in North Europeans, in whom the disequilibrium between the allelic systems defined by the probes and CF is much greater and on a plateau through the genetic region. The different levels of disequilibrium found in the studied populations suggest that D7S399 and D7S23 are both closer to CF than INT1L1, and provide additional information on the origins and homogeneity of the CF defect.     

Morphology and development of the macronuclei of the ciliates Stylonychia mytilus and Euplotes aediculatus

Some stages of macronuclear anlagen development, known from earlier investigations (see Fig. 1), were studied in detail. The results are: a) The giant chromosomes of Stylonychia mytilus are not somatically paired, but are connected end-to-end to form one or a few composite chromosomes. When they later disintegrate, the bands become isolated granules. b) Spectrophotometric measurements show that during the DNA-poor stage which follows the disintegration of the chromosomes, the macronuclear anlagen of Euplotes have a DNA content of 21 c, while the syncaryotic (deriving from syncarya) and hemicaryotic (deriving from haploid hemicarya) anlagen of Stylonychia have the DNA content of diploid micronuclei (2c). Nevertheless the syncaryotic anlagen of Stylonychia and Euplotes initially develop two nucleoli at the end of this stage, the hemicaryotic anlagen of Stylonychia only one. From this it is concluded that the genes of one giant chromosome band stay together in one granule, c) Labeled DNA from the giant chromosomes which remains in the anlagen during the DNA-poor stage is distributed approximately equally to the daughter nuclei during the first few fissions of the exconjugants.-Autoradiographic experiments showed that the DNA of the macronuclei of Stylonychia that is duplicated at one time in a replication band is not duplicated simultaneously during the next DNA-duplication. The DNA duplications during the second polyploidization stage of the macronuclear anlagen development are exceptions, because the mixing of the macronuclear DNA which occurs before every fission does not occur during the second polyploidization stage.—The pseudomicronuclei which sometimes are formed from the macronuclei in emicronucleated strains of Stylonychia contain numerous elements which are much smaller than the chromosomes.—The macronucleus of Stylonychia is very insensitive to irradiation with X-rays.—The results lead to the following hypothesis: The macronuclei of the two hypotrich ciliates contain unconnected chromomeres or small aggregates which are distributed at random to the two daughter nuclei during the divisions.Research supported by the Deutsche Forschungsgemeinschaft.     

Electron microscopic studies on the macronuclear development in the ciliate Chilodonella uncinata.

The development of macronuclear anlage in the ciliate Chilodonella uncinata has been studied through electron microscopy. The ultrastructure of macro- and micronuclei is described for comparison. During the first stage of development, when the DNA content of the macronuclear anage increases from 2 C to 32 C, chromosomes are visible as distinct osmiophilic bodies inside the anlage. At the end of the initial polyploidization phase, the chromosomes despiralize, giving rise to long filamentous structures 25 to 50 nm in diameter. The latter show a singular banding pattern, with dense bands 12 to 25 nm thick alternating regularly with less dense interbands. Such an organization has not yet been observed in any other type of nucleus. These filamentous structures have been interpreted as highly despiralized oligotenic chromosomes. During the final stage of macronuclear development, these structures condense into thin chromatin strands and small dense granules; the number of granules increases progressively as the chromatin strands disappear. These small granules very likely fuse amongst themselves to form the chromatin granules of the vegetative macronucleus. No evidence has yet been found for a fragmentation of chromatin in this ciliate, but this problem needs further study. The old macronucleus maintains a normal ultrastructure until a late stage of development of the macronuclear anlage, becoming pycnotic only towards the very end of the latter process.     

Rate of phenotypic assortment in Tetrahymena thermophila

During vegetative, asexual reproduction in heterozygous Tetrahymena 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 R_f values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of R_f. 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, Inc.