The structure of Tetrahymena pyriformis mitochondrial DNA. II. The complex structure of strain GL mitochondrial DNA.

1. Isolated mtDNA from Tetrahymena pyriformis strain GL is a linear duplex molecule with an average molecular weight of 32.6 - 10(6) and without internal gaps or breaks. Denaturation of this DNA results in single strands with a duplex hairpin at one end. The length of this hairpin varies between 0 and 5 micrometer within one preparation. 2. Uder renaturation conditions the single strands with hairpins are able to circularize in two ways, depending on the length of the hairpin. Circularization is also observed after partial digestion with exonuclease III of native strain GL mtDNA. 3. All these data fit a model (see Fig.2) in which the DNA is heterogeneous in length at both ends. At the left end a 10-micrometer duplication-inversion is present; part of this duplication-inversion is complementary to a region at the right end of the molecule. 4. The analogy between the structural peculiarities of strain GL mtDNA and of some linear viral DNAs is stressed.     

Mitochondrial telomeres: surprising diversity of repeated telomeric DNA sequences among six species of Tetrahymena

The DNA sequences at the ends of the linear mtDNA of 6 species of Tetrahymena encompassing 13 strains were determined. All the strains have variable numbers of a tandemly repeated DNA sequence, 31 bp to 53 bp in size, at their mtDNA termini. Based upon the size and nucleotide sequence of the terminal repeats, the telomeres can be separated into four classes. T. pigmentosa, hyperangularis, and hegewischi have different telomeric repeats on the two ends of their mtDNAs. The only conserved feature of the mtDNA termini is the presence of tandem repeats. The function of the repeats might be to promote unequal crossing over during recombination, thereby overcoming the problem of telomere replication for these linear DNAs.     

Conservation of the sequence and position of the ribosomal RNA genes in Tetrahymena pyriformis mitochondrial DNA.

1. We have done cross-hybridizations between the mitochondrial ribosomal RNAs and DNAs from strains ST and PP of Tetrahymena pyriformis. DNA . ribosomal RNA hybrid formation can be completely prevented by an excess of the heterologous ribosomal RNA and the heterologous hybrids melt 6 degrees C below the homologous hybrids. This shows that the ribosomal RNA cistrons can account for the 5% cross-hybridization previously observed between the mtDNAs of strains PP and ST (Goldbach et al. (1977) Biochim. Biophys. Acta 477, 37--50). 2. By electron microscopy of DNA . ribosomal RNA hybrids we have determined the position of the ribosomal RNA cistrons on the mtDNA of strain GL, a mtDNA which we have shown to contain a sub-terminal 1 micron duplication-inversion and a terminal palindrome at one end which varies in length from 0 to 5 micron and which includes the 1 micron duplication-inversion (Arnberg et al. (1977) Biochim. Biophys. Acta 477, 51--69). The 21 S ribosomal RNA cistron overlaps the 1 micron duplication-inversion and as a result two or three cistrons are present, depending on the size of the terminal palindrome. Only one 14 S ribosomal RNA cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron and with the same polarity as this cistron. 3. We conclude from these results and those in the preceding paper that the sequence of the ribosomal RNAs and the position of the ribosomal RNA genes in the mtDNA is strongly conserved in Tetrahymena. Possible reasons for the duplication of 21-S ribosomal RNA genes and the terminal heterogeneity of Tetrahymena mtDNA are discussed.     

Replication of the linear mitochondrial DNA of Tetrahymena pyriformis.

1. Electron micrographs of the linear mtDNA from Tetrahymena pyriformis strain GL show linear molecules with a duplex 'eye' of variable size in the middle. This indicates that replication of this DNA starts near the middle of the molecule and proceeds bidirectionally to the ends, as previously shown for the mtDNA of strain ST (Arnberg, A.C., Van Bruggen, E.F.J., Clegg, R.A., Upholt, W.B. and Borst, P. (1974) Biochim. Biophys. Acta 361, 266-276). The mtDNAs of these two strains have little base sequence homology beyond the ribosomal RNA cistron (Goldbach, R.W., Bollen-De Boer, J.E., Van Bruggen, E.F.J. and Borst, P. (1978) Biochim. Biophys. Acta 521, 187-197). 2. Electron micrographs of mtDNA from strain ST, spread under non-denaturing conditions, contain only molecules with fully duplex ends. mtDNA spread under conditions of early denaturation contains duplex loops on one end (40% of all molecules) or both ends (37%). The loops are stable to partial denaturation and vary in size from 0.15 to approximately 1.0 micron, most loops measuring 0.25--0.40 micron. No loops are formed with single-stranded DNA under analogous conditions and we conclude from this result that loop formation is based on the presence of straight, rather than inverted, duplications near the ends. 3. When full-length 3H-labelled mtDNA from strain ST, 32P-labelled at the 5'-termini with T4 polynucleotide kinase, was sedimented in alkaline sucrose gradients, greater than 70% of the 3H and less than 30% of the 32P cosedimented with full-length molecules; the remaining 32P sedimented heterogeneously and predominantly with the DNA less than 10% the size of intact single strands. Brief incubations of full-length mtDNA with DNA polymerase I from Escherichia coli and labelled dNTPs at 15 degrees C did not lead to preferential labelling of terminal EcoRI fragments of the DNA. From these results we infer that the DNA contains nicks or gaps near the termini and that these are not bordered by free 3'-OH groups. 4. A model is presented in which straight sequence repetitions at the termini of Tetrahymena pyriformis mtDNA are involved in the later stages of replication. This model can also account for the pronounced terminal heterogeneity previously observed in this DNA.     

Mapping of mitochondrial DNA of individual sheep and goats: Rapid evolution in the D loop region

Mitochondrial DNA (mtDNA) from sheep and goat was compared by restriction endonuclease analysis and heteroduplex mapping in the electron microscope. The fragment patterns produced by endonuclease Hae III from three individual sheep and two goat mtDNAs all differed from each other. The three sheep mtDNAs had identical Eco RI and Hind III fragments, but the two goat mtDNA patterns differed from each other as well as from sheep mtDNA. We estimate that each sheep mtDNA differs from each other by 0.5–1% of its nucleotide sequences, the two goat mtDNAs by 1–2%, and there is a 6–11% sequence difference between sheep and goat mtDNAs. We have mapped the Eco RI and Hind III sites of goat and sheep mtDNA and determined the positions of the D loop, which marks the replication origin, relative to the restriction map. The D loops are at homologous positions on the mtDNAs from both species, but the goat D loop is only 75% as long as the sheep D loop. Regions with a high degree of sequence divergence occur at both ends of the D loop. We suggest that a duplication of about 150 base pairs has occurred in the region where the sheep and goat D loops differ in length. We discuss mtDNA evolution in terms of divergence of isolated “mitochondrial DNA clones.”     

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST.

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.     

Nuclear-mitochondrial epistasis and drosophila aging: introgression of Drosophila simulans mtDNA modifies longevity in D. melanogaster nuclear backgrounds

Under the mitochondrial theory of aging, physiological decline with age results from the accumulated cellular damage produced by reactive oxygen species generated during electron transport in the mitochondrion. A large body of literature has documented age-specific declines in mitochondrial function that are consistent with this theory, but relatively few studies have been able to distinguish cause from consequence in the association between mitochondrial function and aging. Since mitochondrial function is jointly encoded by mitochondrial (mtDNA) and nuclear genes, the mitochondrial genetics of aging should be controlled by variation in (1) mtDNA, (2) nuclear genes, or (3) nuclear-mtDNA interactions. The goal of this study was to assess the relative contributions of these factors in causing variation in Drosophila longevity. We compared strains of flies carrying mtDNAs with varying levels of divergence: two strains from Zimbabwe (<20 bp substitutions between mtDNAs), strains from Crete and the United States (approximately 20-40 bp substitutions between mtDNAs), and introgression strains of Drosophila melanogaster carrying mtDNA from Drosophila simulans in a D. melanogaster Oregon-R chromosomal background (>500 silent and 80 amino acid substitutions between these mtDNAs). Longevity was studied in reciprocal cross genotypes between pairs of these strains to test for cytoplasmic (mtDNA) factors affecting aging. The intrapopulation crosses between Zimbabwe strains show no difference in longevity between mtDNAs; the interpopulation crosses between Crete and the United States show subtle but significant differences in longevity; and the interspecific introgression lines showed very significant differences between mtDNAs. However, the genotypes carrying the D. simulans mtDNA were not consistently short-lived, as might be predicted from the disruption of nuclear-mitochondrial coadaptation. Rather, the interspecific mtDNA strains showed a wide range of variation that flanked the longevities seen between intraspecific mtDNAs, resulting in very significant nuclear x mtDNA epistatic interaction effects. These results suggest that even "defective" mtDNA haplotypes could extend longevity in different nuclear allelic backgrounds, which could account for the variable effects attributable to mtDNA haplogroups in human aging.     

Conservation of sequences adjacent to the telomeric C4A2 repeats of ciliate macronuclear ribosomal RNA gene molecules.

We sequenced and compared the telomeric regions of linear rDNAs from vegetative macronuclei of several ciliates in the suborder Tetrahymenina. All telomeres consisted of tandemly repeated C4A2 sequences, including the 5' telomere of the 11 kb rDNA from developing macronuclei of Tetrahymena thermophila. Our sequence of the 11 kb 5' telomeric region shows that each one of a previously described pair of inverted repeats flanking the micronuclear rDNA (Yao et al., Mol. Cell. Biol. 5: 1260-1267, 1985) is 29 bp away from the positions to which telomeric C4A2 repeats are joined to the ends of excised 11 kb rDNA. In general we found that the macronuclear rDNA sequences adjacent to C4A2 repeats are not highly conserved. However, in the non-palindromic rDNA of Glaucoma, we identified a single copy of a conserved sequence, repeated in inverted orientation in Tetrahymena spp., which all form palindromic rDNAs. We propose that this sequence is required for a step in rDNA excision common to both Tetrahymena and Glaucoma.     

Cloning yeast telomeres on linear plasmid vectors

We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hairpin terminus was unable to stabilize a linear plasmid in yeast. The fact that yeast can recognize and use DNA ends from the distantly related organism Tetrahymena suggests that the structural features required for telomere replication and resolution have been highly conserved in evolution. The linear plasmid was used as a vector to clone chromosomal telomeres from yeast. One Tetrahymena end was removed by restriction digestion, and yeast fragments that could function as an end on a linear plasmid were selected. Restriction mapping and hybridization analysis demonstrated that these fragments were yeast telomeres, and suggested that all yeast chromosomes might have a common telomere sequence. Yeast telomeres appear to be similar in structure to the rDNA of Tetrahymena, in which specific nicks or gaps are present within a simple repeated sequence near the terminus of the DNA.     

Tetrahymena telomerase RNA levels increase during macronuclear development.

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000-40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2- to 5-fold at 9-15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells.     

Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature

Mitochondrial DNAs from ten wild-type Neurospora crassa, Neurospora intermedia, and Neurospora sitophila strains collected from different geographical areas were screened for structural variations by restriction enzyme analysis. The different mtDNAs show much greater structural diversity, both within and among species, than had been apparent from previous studies of mtDNA from laboratory N. crassa strains. The mtDNAs range in size from 60 to 73 kb, and both the smallest and largest mtDNAs are found in N. crassa strains. In addition, four strains contain intramitochondrial plasmid DNAs that do not hybridize with the standard mtDNA. All of the mtDNA species have a basically similar organization. A 25-kb region that includes the rRNA genes and most tRNA genes shows very strong conservation of restriction sites in all strains. The 2.3-kb intron found in the large rRNA gene in standard N. crassa mtDNAs is present in all strains examined, including N. intermedia and N. sitophila strains. The size differences between the different mtDNAs are due to insertions or deletions that occur outside of the rRNA-tRNA region. Restriction enzyme and heteroduplex mapping suggest that four of these insertions are optional introns in the gene encoding cytochrome oxidase subunit I. Mitochondrial DNAs from different wild-type strains contain zero, one, three, or four of these introns.     

Microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs

Telomeres are essential structures that stabilize the ends of eukaryotic chromosomes and allow complete replication of linear DNA molecules. We examined the structure and replication of telomeres by observing the fate of the linear extrachromosomal rDNA of Tetrahymena after injection into unfertilized Xenopus eggs. The rDNA replicated efficiently as a linear extrachromosomal molecule, increasing in mass 30-50-fold by 15-20 h after injection. In addition, the molecules increased in length by addition of up to several kilobases of DNA to their termini. Sequence analysis demonstrated that the added DNA bore no resemblance to known telomeres. The junction between the rDNA and added DNA was apparently random, indicating that the addition reaction did not involve a site-specific recombination or integration event. Surprisingly, Southern blot analysis showed that the added DNA did not derive from Xenopus DNA, but rather from co-purifying and therefore co-injected Tetrahymena DNA. The nonspecific ligation of random DNA fragments to the rDNA termini suggests that microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.     

Preparation and melting of single strand circular DNA loops.

A method for preparation of single strand DNA circles of almost arbitrary sequence is described. By ligating two sticky ended hairpins together a linear duplex is formed, closed at both ends by single stranded loops. The melting characteristics of such loops are investigated using optical absorbance and NMR. It is shown by comparison with the corresponding linear sequence (closed circle minus the end loops) that the effects of end fraying and the strand concentration dependence of the melting temperature are eliminated in the circular form. Over the concentration range examined (0.5 to 2.0 micromolar strands), the circular DNA has a monophasic melting curve, while the linear duplex is biphasic, probably due to hairpin formation. Since effects of duplex to single strands dissociation do not contribute to melting of the circular molecules (dumbells), these DNAs present a realistic experimental model for examining local thermal stability in DNA.     

Tandemly repeated hexanucleotide at Tetrahymena rDNA free end is generated from a single copy during development

The linear extrachromosomal ribosomal DNA of Tetrahymena is generated from a single integrated copy during macronuclear development. The free ends of this extrachromosomal gene contain 20-70 tandem repeats of the hexanucleotide CCCCAA. We have determined the nucleotide sequence at the same (3') end of the single, integrated micronuclear gene. In contrast to the extrachromosomal gene, only a single CCCCAA sequence was found at this position. The same result was obtained from two independently isolated DNA clones, and was therefore not likely an artifact of cloning. Comparisons of the genomic DNA with the cloned fragment by Southern hybridization also supported this argument. Thus the tandemly repetitive hexanucleotide at the free ends of the extrachromosomal rDNA is not an inherited feature, and must be generated during the development of the macronucleus.     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions.

Electron microscopy of purified full-length linear duplex molecules produced by bleomycin reaction with PM2 DNA revealed low frequencies of closed circular duplex molecules as well as linear duplex molecules with opposed ends (cyclized molecules which have dissociated to yield a gap between the termini). The occurrence of these latter forms indicates that double-strand scissions produced by bleomycin reaction consist of two single-strand scissions which are physically staggered on the complementary strands. Analysis of the temperature dependence for cyclization led to the estimate that an average of 1.7 +/- 0.44 base-pairs (2.6 +/- 0.5 base pairs without base-stacking energies) occur between the staggered breaks. The reassociated termini cannot be ligated with T4 ligase. When PM2 DNA was fragmented at several sites within each molecule, circular duplexes and linear duplexes with opposed ends with a range of sizes from 350 base pairs up to full-length PM2 DNA were observed. Analysis of the frequency distribution of lengths of these fragments indicates that most, if not all, of the specific sites for bleomycin-directed double-strand scissions in PM2 DNA contain representatives of the same two base single-stranded termini.     

mtDNA variation of aboriginal Siberians reveals distinct genetic affinities with Native Americans.

The mtDNA variation of 411 individuals from 10 aboriginal Siberian populations was analyzed in an effort to delineate the relationships between Siberian and Native American populations. All mtDNAs were characterized by PCR amplification and restriction analysis, and a subset of them was characterized by control region sequencing. The resulting data were then compiled with previous mtDNA data from Native Americans and Asians and were used for phylogenetic analyses and sequence divergence estimations. Aboriginal Siberian populations exhibited mtDNAs from three (A, C, and D) of the four haplogroups observed in Native Americans. However, none of the Siberian populations showed mtDNAs from the fourth haplogroup, group B. The presence of group B deletion haplotypes in East Asian and Native American populations but their absence in Siberians raises the possibility that haplogroup B could represent a migratory event distinct from the one(s) which brought group A, C, and D mtDNAs to the Americas. Our findings support the hypothesis that the first humans to move from Siberia to the Americas carried with them a limited number of founding mtDNAs and that the initial migration occurred between 17,000-34,000 years before present.     

A novel class of nucleolar RNAs from Tetrahymena.

We describe a family of at least four nucleolar RNAs (snoRNAs) from the ciliate, Tetrahymena. The snoRNAs are 120-140 nucleotides long, moderately AU-rich and contain no modified nucleotides. Their 5' ends are blocked by a cap of unknown nature. The snoRNAs can be folded into similar secondary structures consisting of two hairpins separated by a single-stranded AU-rich spacer. The sequences and secondary structures show no extensive sequence or secondary structure resemblance to any other small RNAs in the public databases.