Elimination of specific DNA sequences from the somatic nucleus of the ciliate Tetrahymena

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.     

Non-Mendelian, heritable blocks to DNA rearrangement are induced by loading the somatic nucleus of Tetrahymena thermophila with germ line-limited DNA.

Site-specific DNA deletion occurs at thousands of sites within the genome during macronuclear development of Tetrahymena thermophila. These deletion elements are usually not detected in macronuclear chromosomes. We have interfered with the normal deletion of two of these elements, the adjacent M and R elements, by loading vegetative macronuclei with these elements prior to sexual conjugation. Transformed cell lines containing the exogenous M or R element, carried on high-copy-number vectors containing genes encoding rRNA within parental (old) macronuclei, consistently failed to excise chromosomal copies of the M or R element during formation of new macronuclei. Little or no interference with the deletions of adjacent elements or of unlinked elements was observed. The micronucleus (germ line)-limited region of each element was sufficient to inhibit specific DNA deletion. This interference with DNA deletion usually is manifested as a cytoplasmic dominant trait: deletion elements present in the old macronucleus of one partner of a mating pair were sufficient to inhibit deletion occurring in the other partner. Remarkably, the failure to excise these elements became a non-Mendelian, inheritable trait in the next generation and did not require the high copy number of exogenously introduced elements. The introduction of exogenous deletion elements into parental macronuclei provides us with an epigenetic means to establish a heritable pattern of DNA rearrangement.     

A family of moderately repetitive sequences in mouse DNA.

When mouse DNA is digested to completion with restriction endonuclease Eco R1, a distinct band of 1.3 kb segments comprising about 0.5-3% of the genome is observed upon agarose gel electrophoresis. This DNA is not tandemly repeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kb band and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 10(4) times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco R1 band.     

SCAR DNA family is enriched in evolutionarily conserved sequences

The family of DNA sequences tightly associated with the synaptonemal complex (SC), or SCAR DNA family, has earlier been described as a specific family of golden hamster genomic DNA sequences. DNA sequences similar to golden hamster SCAR DNA proved to be widespread in the genomes of some vertebrates. A comparison with a sample of random sequences showed that the SCAR DNA family is enriched in evolutionarily conserved sequences, which correlates with the universal SC morphology and processes occurring in meiotic prophase I.     

The SCAR DNA sequence family is enrichment in the evolution-conserved sequences

Synaptonemal complex (SC) is a specific structure for prophase I of meiosis. Recently we have described synaptonemal complex tightly associated regions of DNA (SCARs DNA) as a particular family of genomic DNA. Now we reveal the evolutionary conservation of SCAR DNA sequences of vertebrates. This data correlates with universal morphology of SCs and similar processes proceed in prophase I of meiosis at representatives of different taxa.     

Reproducible and variable genomic rearrangements occur in the developing somatic nucleus of the ciliate Tetrahymena thermophila.

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.     

Nucleotide sequences of three tRNA genes encoded in Tetrahymena mitochondrial DNA.

Nucleotide sequences of three cloned restriction fragments of Tetrahymena mtDNA which showed hybridization with mitochondrial tRNA have been determined. EcoRI fragment 5 (4.1 kbp) contains the tRNAphe gene sequence with anticodon GAA; Hind III fragment 6 (2.0 kbp) the tRNAhis with anticodon GTG; and EcoRI fragment 7 (1.9 kbp) the tRNAtrp with anticodon TCA. The CCA end is not encoded. All three tRNAs show usual features with common invariant and semi-invariant bases and can be folded into a cloverleaf structure with standard loops and regular base pairs in the stems. However, some minor irregular features are present including several GT pairs and an unmatched TT in the stems, and TCC instead of T psi C. All exhibit high G+C contents (about 50%); in contrast, the flanking regions are extremely A+T rich (about 80%). Several short coding frames can be deduced in these sequences, but their significance is not known.     

Rearrangement of repeated DNA sequences during development of macronucleus in Tetrahymena thermophila.

Three clones of non-repetitive sequences and six clones containing repetitive sequences were obtained from micronuclear DNA of Tetrahymena thermophila. All the non-repetitive and three repetitive sequences had the same organization in micro- and macronuclear DNAs as revealed by blot hybridization. On the other hand, the remaining three clones with repetitive sequences had apparently different organization in the two nuclear DNAs. All these repetitive sequences showed a smear on the blot in addition to a number of discrete bands when micronuclear DNA was digested with EcoR I. In macronuclear DNAs, these sequences invariably became one or two bands and the smear disappeared. We conclude that, when a macronucleus develops from a micronucleus, the non-repetitive sequences amplify by more than 20 times with relatively few rearrangement, whereas some selected portions of repeated and/or repeat-contiguous sequences are amplified with rather extensive reorganization.     

A ubiquitous family of repeated DNA sequences in the human genome

Renatured DNA from human and many other eukaryotes is known to contain 300-nucleotide duplex regions formed from renatured repeated sequences. These short repeated DNA sequences are widely believed to be interspersed with single copy DNA sequences. In this work we show that at least half of these 300-nucleotide duplexes share a cleavage site for the restriction enzyme AluI. This site is located 170 nucleotides from one end. This Alu family of repeated sequences makes up at least 3% of the genome and is present in several hundred thousand copies.Inverted repeated sequences are also known to contain a short 300-nucleotide duplex region. We find that at least half of the 300-nucleotide duplex regions in inverted repeated sequences also have an AluI restriction site located 170 nucleotides from one end.By driven renaturation techniques, the Alu family is shown to be distributed over a minimum range of 30% to 60% of the genome. (The breadth of this range reflects the presence of inverted repeated sequences which, in part, include the Alu family.) These findings imply that the interspersion pattern of repeated and single copy sequences in human DNA is largely dominated by one family of repeated sequences.     

A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila.

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.     

A family of repetitive palindromic sequences found in Neurospora mitochondrial DNA is also found in a mitochondrial plasmid DNA

Neurospora mtDNA contains a repetitive, 18 nucleotide palindromic sequence (5'-CCCTGCAGTACTGCAGGG-3') that contains two closely spaced PstI sites (CTGCAG) in the arms of the palindrome (Yin, S., Heckman, J., and RajBhandary, U. L. (1981) Cell 26, 325-332). In the present study, DNA sequence analysis was carried out to determine whether PstI palindromes are present in an apparently distinct genetic element, the 3.6-kilobase mitochondrial plasmid from Neurospora crassa strain Mauriceville-1c (FGSC 2225). The plasmid contains a cluster of closely spaced PstI sites extending over a 0.4-kilobase region (Collins, R. A., Stohl, L. L., Cole, M. D., and Lambowitz, A. M. (1981) Cell 24, 443-452). The DNA sequence shows that the cluster consists of eight PstI sites organized in five palindromic elements. Two of the elements are identical with the canonical sequence found in mtDNA, whereas the remaining three elements differ from the canonical sequence by a few nucleotides. The occurrence of the PstI palindromes in two otherwise unrelated DNA species is consistent with the hypothesis that they are related to mobile DNA sequences that either propagate or were once capable of propagating within mitochondria.     

A new family of interspersed repetitive DNA sequences in the mouse genome

Repetitive DNA sequences near immunoglobulin genes in the mouse genome (Steinmetz et al., 1980a,b) were characterized by restriction mapping and hybridization. Six sequences were determined that turned out to belong to a new family of dispersed repetitive DNA. From the sequences, which are called R1 to R6, a 475 base-pair consensus sequence was derived. The R family is clearly distinct from the mouse B1 family (Krayev et al., 1980). According to saturation hybridization experiments, there are about 100,000 R sequences per haploid genome, and they are probably distributed throughout the genome. The individual R sequences have an average divergence from the consensus sequence of 12.5%, which is largely due to point mutations and, among those, to transitions. Some R sequences are severly truncated. The R sequences extend into A-rich sequences and are flanked by short direct repeats. Also, two large insertions in the R2 sequence are flanked by direct repeats. In the neighbourhood of and within R sequences, stretches of DNA have been identified that are homologous to parts of small nuclear RNA sequences. Mouse satellite DNA-like sequences and members of the B1 family were also found in close proximity to the R sequences. The dispersion of R sequences within the mouse genome may be a consequence of transposition events. The possible role of the R sequences in recombination and/or gene conversion processes is discussed.     

Comparison of the sequences of macro- and micronuclear DNA of Tetrahymena pyriformis

Macro- and micronuclei were isolated from Tetrahymena pyriformis (Syngen 1, strain WH-6) and their DNAs compared by isopycnic centrifugation in neutral and alkaline CsCl, by analysis of thermal denaturation properties and by molecular hybridization. Unlike the situation observed in Stylonychia the buoyant densities and thermal denaturation patterns of Tetrahymena macro- and micronuclear DNAs were virtually identical—the only observable differences bordering on the limits of resolution of these techniques. DNA was isolated from the two nuclei which had been labelled with different radioactive isotopes (i.e. ¹⁴C-thymidine and ³H-thymidine), and the renaturation kinetics of mixtures of macro- and micronuclear DNA were examined using a single-strand specific deoxyribonuclease (S₁). Renaturation kinetics obtained using varying ratios of macro- and micronuclear DNA suggested that 80–90% of the sequences present in micronuclei were present in similar amounts in macronuclei. However, careful analyses of the renaturation kinetics indicate that approximately 10–20% of the sequences found in micronuclei are probably absent in macronuclei, and that most of these sequences are probably moderately repetitive (100 copies per genome or less). These findings place severe constraint on possible models concerning the structure of the Tetrahymena macronucleus, and are very different from the situation observed in Stylonychia where it has been suggested that only a small percentage of the sequences in micronuclei are present in significant amounts in macronuclei. Nonetheless, these results along with those in Stylonychia can be taken as an indication that the loss or under-replication of some DNA sequences accompanies macronuclear differentiation in ciliates.