Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested. A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T. thermophila. After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants. Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions. The circular plasmid carrying an inverted repeat of T. thermophila telomeres could be linearized and processed in vivo.     

Tetrahymena micronuclear sequences that function as telomeres in yeast.

We explored the ability of S. cerevisiae to utilize heterologous DNA sequences as telomeres by cloning germline (micronuclear) DNA from Tetrahymena thermophila on a linear yeast plasmid that selects for telomere function. The only Tetrahymena sequences that functioned in this assay were (C4A2)n repeats. Moreover, these repeats did not have to be derived from Tetrahymena telomeres, although we show that micronuclear telomeres (like macronuclear telomeres) of Tetrahymena terminate in (C4A2)n repeats. Chromosome-internal restriction fragments carrying (C4A2)n repeats also stabilized linear plasmids and were elongated by yeast telomeric repeats. In one case, the C4A2 repeat tract was approximately 1.5 kb from the end of the genomic Tetrahymena DNA fragment that was cloned, but this 1.5 kb of DNA was missing from the linear plasmid. Thus, yeast can utilize internally located tracts of telomere-like sequences, after the distal DNA is removed. The data provide an example of broken chromo-some healing, and underscore the importance of the telomeric repeat structure for recognition of functional telomeric DNA in vivo.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences. Received: 13 January 1998 / Accepted: 10 June 1998     

In VivoAddition of Telomeric Repeats to Foreign DNA Generates Extrachromosomal DNAs in the Taxol-Producing FungusPestalotiopsis microspora

Transformation of the taxol-producing filamentous fungusPestalotiopsis microsporawith a plasmid containing the bacterial hygromycin resistance gene fused toAspergillusregulatory sequences resulted in thein vivoformation of extrachromosomal DNAs with telomeric repeats in the majority of transformants. Repeats of the telomeric sequence 5′-TTAGGG-3′ were appended to nontelomeric transforming DNA termini. No fungal sequences other than telomeric repeats were detected in extrachromosomal DNAs. Transformants contained three to six different sizes or conformational forms of extrachromosomal DNAs. The DNAs showed no change in size or internal structure during 6 months of growth with selection, but were lost after 20 days of growth without selection. Transformation of wild-typeP. microsporawith a PCR-amplified extrachromosomal DNA having terminal telomeric repeats produced up to 50-fold more transformants than the original transformation vector. The addition of telomeric repeats to foreign DNA is unusual among fungi and may have important adaptive or developmental implications.     

Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae.

We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation. The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules. The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA. The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule. Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA. The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase. The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division.     

In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum

Histoplasma capsulatum is a dimorphic pathogenic fungus that is a major cause of respiratory and systemic mycosis. We previously developed a transformation system for Histoplasma and demonstrated chromosomal integration of transforming plasmid sequences. In this study, we describe another Histoplasma mechanism for maintaining transforming DNA i.e. the generation of modified, multicopy linear plasmids carrying DNA from the transforming Escherichia coli plasmid. Under selective conditions, these linear plasmids were stable and capable of retransforming Histoplasma without further modification. In vivo modification of the transforming DNA included duplication of plasmid sequence and telomeric addition at the termini of linear DNA. Apparently Histoplasma telomerase, like that of other organisms such as humans and Tetrahymena, is able to act on non-telomeric substrates. The terminus of a Histoplasma linear plasmid was cloned and shown to contain multiple repeats of GGGTTA, the telomeric repeat unit also found in vertebrates, trypanosomes, and slime moulds.     

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats.     

Chromosome termini of the monocot plant Othocallis siberica are maintained by telomerase, which specifically synthesises vertebrate-type telomere sequences

Lack of Arabidopsis-type T3AG3 telomere sequences has recently been reported for the majority of investigated taxa of the monocot order Asparagales. In order to investigate this phenomenon in more detail, we conducted extensive cytogenetic and molecular analyses of the telomeres in Othocallis siberica, a member of this order. Terminal restriction fragment analysis together with Bal31 exonuclease assay showed that chromosome termini in O. siberica are formed by long stretches (more than 10 kbp) of vertebrate-type T2AG3 repeats. In addition, telomerase activity specifically synthesising (T2AG3)n sequence was detected in O. siberica protein extracts by telomerase repeat amplification protocol (TRAP). Fluorescence in situ hybridisation (FISH) revealed the presence of the vertebrate-type T2AG3 telomere sequences at all chromosome termini and at a few additional regions of O. siberica chromosomes, whereas Arabidopsis-type T3AG3 DNA and peptide nucleic acid (PNA) probes did not hybridise to chromosomes of Othocallis, except for polymorphic blocks in chromosomes 2 (interstitial) and 4 (terminal). These interstitial/terminal regions are apparently composed of large blocks of (T2AG3)n and (T3AG3)n DNA and represent a unique example of interspersion of two types of telomeric repeats within one genome. This may be a reflection of the recent evolutionary switch from Arabidopsis- to vertebrate-type telomeric repeats in this plant group.     

Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.     

Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila.

Telomeric DNA of Tetrahymena thermophila consists of a long stretch of (TTGGGG)n double-stranded repeats with a single-stranded (TTGGGG)2 3' overhang at the end of the chromosome. We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3' telomeric repeat overhang. This protein is called TEP (telomere end-binding protein). A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold. Changing two G's to C's in the TTGGGG repeats totally abolishes binding. However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding. A duplex structure adjacent to the 3' overhang is required for binding, although the duplex need not contain telomeric repeats. TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences. TEP has a greatly reduced affinity for RNA substrates. The copy number of TEP is at least 2 x 10(4) per cell, and it is present under different conditions of cell growth and development, although its level varies. UV cross-linking experiments show that TEP has an apparent molecular mass of approximately 65 kDa. Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.     

Recognition and elongation of telomeres by telomerase

Telomeres stabilize chromosomal ends and allow their complete replication in vivo. In diverse eukaryotes, the essential telomeric DNA sequence consists of variable numbers of tandem repeats of simple, G + C rich sequences, with a strong strand bias of G residues on the strand oriented 5' to 3' toward the chromosomal terminus. This strand forms a protruding 3' over-hang at the chromosomal terminus in three different eukaryotes analyzed. Analysis of yeast and protozoan telomeres showed that telomeres are dynamic structures in vivo, being acted on by shortening and lengthening activities. We previously identified and partially purified an enzymatic activity, telomere terminal transferase, or telomerase, from the ciliate Tetrahymena. Telomerase is a ribonucleoprotein enzyme with essential RNA and protein components. This activity adds repeats of the Tetrahymena telomeric sequence, TTGGGG, onto the 3' end of a single-stranded DNA primer consisting of a few repeats of the G-rich strand of known telomeric, and telomere-like, sequences. The shortest oligonucleotide active as a primer was the decamer G4T2G4. Structural analysis of synthetic DNA oligonucleotides that are active as primers showed that they all formed discrete intramolecular foldback structures at temperatures below 40 degrees C. Addition of TTGGGG repeats occurs one nucleotide at a time by de novo synthesis, which is not templated by the DNA primer. Up to 8000 nucleotides of G4T2 repeats were added to the primer in vitro. We discuss the implications of this finding for regulation of telomerase in vivo and a model for telomere elongation by telomerase.     

Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase.

Telomeric DNA at the ends of chromosomes consist of short, tandem repeat sequences. The telomeres of Paramecium tetraurelia are made up of variable repeats, whereas Paramecium caudatum telomeric repeats are largely invariant. To investigate variable repeat synthesis in P. tetraurelia, mutated telomerase RNA genes were expressed in vivo. We demonstrate that the P. caudatum telomerase RNA can participate in telomere synthesis when expressed in the P. tetraurelia macronucleus, despite 24% primary sequence divergence of the RNAs between the two species. De novo telomeric repeats from transformants indicate that P. tetraurelia telomerase fidelity is dramatically affected by template substitutions and that misincorporation at a single templating position is likely to account for the majority of P. tetraurelia telomeric DNA variability. Furthermore, we show that fidelity is not solely a function of the RNA moiety, as the P. caudatum telomerase RNA does not impart high fidelity to the chimeric enzyme.     

Progressive rearrangement of telomeric sequences added to both the ITR ends of the yeast linear pGKL plasmid

Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. InSaccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid) which carried the host telomeric repeats TG_1–3 of 300–350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat), suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG_1–3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids. Published: February 17, 2003     

S1 nuclease sensitivity of a double-stranded telomeric DNA sequence.

We examined structural properties of poly d(C4A2).d(T2G4), the telomeric DNA sequence of the ciliated protozoan Tetrahymena. Under conditions of high negative supercoiling, poly d(C4A2).d(T2G4) inserted in a circular plasmid vector was preferentially sensitive to digestion with S1 nuclease. Only the C4A2 strand was sensitive to first-strand S1 cutting, with a markedly skewed pattern of hypersensitive sites in tracts of either 46 or 7 tandem repeats. Linear poly d(C4A2).(T2G4) showed no preferential S1 sensitivity, no circular dichroism spectra indicative of a Z-DNA conformation, no unusual Tm, and no unusual migration in polyacrylamide gel electrophoresis. The S1 nuclease sensitivity properties are consistent with a model proposed previously for supercoiled poly d(CT).d(AG) (Pulleyblank et al., Cell 42:271-280, 1985), consisting of a double-stranded, protonated, right-handed underwound helix. We propose that this structure is shared by related telomeric sequences and may play a role in their biological recognition.     

In vivo rearrangement of foreign DNA by Fusarium oxysporum produces linear self-replicating plasmids.

Particular combinations of fungal strains and transformation vectors allow for fungal rearrangement of normally integrative plasmids, resulting in the creation of linear self-replicating plasmids in Fusarium oxysporum. The rearrangement results in the addition of fungal DNA, including telomere consensus sequences, to plasmid termini. The mechanism by which this rearrangement occurs is unclear, but it has similarities to extrachromosomal gene amplification. A DNA fragment which allows for linear autonomous replication upon reintroduction to the fungus was subcloned and sequenced. This DNA sequence contains the repeated telomeric sequence TTAGGG flanked by a region of twofold symmetry consisting primarily of pUC12 DNA. Isolation and identification of this sequence is the first step toward development of vectors that function as artificial chromosomes in filamentous fungi. This sequence was shown to promote autonomous replication and enhance transformation in several strains of F. oxysporum, Nectria haematococca, and Cryphonectria parasitica.     

The interaction of cisplatin with a human telomeric DNA sequence containing seventeen tandem repeats

The anti-tumour drug, cisplatin, preferentially forms adducts at G-rich DNA sequences. Telomeres are found at the ends of chromosomes and, in humans, contain the repeated DNA sequence (GGGTTA)_n that is expected to be targeted by cisplatin. Using a plasmid clone with 17 tandem telomeric repeats, (GGGTTA)₁₇, the DNA sequence specificity of cisplatin was investigated utilising the linear amplification procedure that pin-pointed the precise sites of cisplatin adduct formation. This procedure used a fluorescently labelled primer and capillary electrophoresis with laser-induced fluorescence detection to determine the DNA sequence specificity of cisplatin. This technique provided a very accurate analysis of cisplatin-DNA adduct formation in a long telomeric repeat DNA sequence. The DNA sequence specificity of cisplatin in a long telomeric tandem repeat has not been previously reported. The results indicated that the 3′-end of the G-rich strand of the telomeric repeat was preferentially damaged by cisplatin and this suggests that the telomeric DNA repeat has an unusual conformation.     

Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina

We have developed an improved transformation procedure for the filamentous fungus Podospora anserina. This procedure is based on the observation that a heat shock at an elevated temperature (48 degrees C) improves the competence of P. anserina protoplasts for transformation 5- to 10-fold. This is observable only if the heat shock is applied before the addition of transforming DNA. An increase in competence is observed immediately after the heat shock, and heat-shocked cells are still competent after 20-30 min. The mechanism by which heat shock improves competence remains unclear. The modified transformation procedure gives as many as 200-500 stable transformants per microgram of plasmid DNA containing the P. anserina ura5 gene. This should allow direct cloning of P. anserina genes from a cosmid library.     

Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability

After selection for migration into the nucleus, a cytoplasmic yeast linear plasmid bearing an inverted terminal repeat (ITRs) at each end replicates in Saccharomyces cerevisiae in a linear form, called pTLU, which carries host telomeric repeats (TG(1-3))(n) of about 300-350 bp added to the ITR ends. We previously showed that the nucleotide composition of the added telomeric sequences varied among individual pTLU isolates, while those on the two ends of any given pTLU were always identical. The telomeric sequences of pTLU remained unchanged over numbers of cell generations when cells were selected for expression of the plasmid-borne nuclear marker. We report here that progressive alterations in telomeric sequences can be detected in cells which are grown under non-selective conditions. Surprisingly, in any given molecule, the telomeric alterations occur exclusively on one side, either the left or the right end, while the sequence at the opposite end remained identical to the original, suggesting a difference in the mode of DNA replication between the plasmid ends. These alterations occur over a broad area extending from the termini of telomeres to nucleotides near the junction between the telomeric sequences and the pTLU-ITR, implying that the plasmid ends undergo successive rounds of extension and contraction. Clonal analysis under non-selective conditions indicated that the alterations in telomeric sequences are generally associated with extreme instability of the pTLU plasmid.     

Stability of telomeric G-quadruplexes

In most eukaryotes, telomeric DNA consists of repeats of a short motif that includes consecutive guanines and may hence fold into G-quadruplexes. Budding yeasts have telomeres composed of longer repeats and show variation in the degree of repeat homogeneity. Although telomeric sequences from several organisms have been shown to fold into G-quadruplexes in vitro, surprisingly, no study has been dedicated to the comparison of G-quadruplex folding and stability of known telomeric sequences. Furthermore, to our knowledge, folding of yeast telomeric sequences into intramolecular G-quadruplexes has never been investigated. Using biophysical and biochemical methods, we studied sequences mimicking about four repetitions of telomeric motifs from a variety of organisms, including yeasts, with the aim of comparing the G-quadruplex folding potential of telomeric sequences among eukaryotes. G-quadruplex folding did not appear to be a conserved feature among yeast telomeric sequences. By contrast, all known telomeric sequences from eukaryotes other than yeasts folded into G-quadruplexes. Nevertheless, while G(3)T(1-4)A repeats (found in a variety of organisms) and G(4)T(2,4) repeats (found in ciliates) folded into stable G-quadruplexes, G-quadruplexes formed by repetitions of G(2)T(2)A and G(2)CT(2)A motifs (found in many insects and in nematodes, respectively) appeared to be in equilibrium with non-G-quadruplex structures (likely hairpin-duplexes).